Specific binding proteins and uses thereof

ABSTRACT

The present invention relates to specific binding members, particularly antibodies and fragments thereof, which bind to EGFR on tumor cells that overexpress EGFR, and on tumor cells that express the truncated version of the EGFR receptor, de2-7 EGF. In particular, the epitope recognized by the specific binding members, particularly antibodies and fragments thereof, is enhanced or evident upon aberrant post-translational modification. These specific binding members are useful in the diagnosis and treatment of cancer. The binding members of the present invention may also be used in therapy in combination with chemotherapeutics or anti-cancer agents and/or with other antibodies or fragments thereof.

RELATED APPLICATION DATA

The present application is a continuation-in-part of and claims priorityto co-pending U.S. patent application Ser. Nos. 12/388,504, filed Feb.18, 2009; and Ser. No. 12/317,683, filed Dec. 23, 2008, each of whichclaim priority to U.S. patent application Ser. No. 10/145,598, filed May13, 2002 (now U.S. Pat. No. 7,589,180, issued Sep. 15, 2009), which is anonprovisional of and claims priority to each of U.S. Provisional PatentApplication Nos. 60/290,410, filed May 11, 2001; 60/326,019, filed Sep.28, 2001; and 60/342,258, filed Dec. 21, 2001; each of which isincorporated by reference herein in its entirety. The presentapplication also incorporates by reference in its entirety thedisclosures of International Patent Application No. PCT/US02/15185,filed May 13, 2002 (Published as WO 02/092771 on Nov. 21, 2002);International Patent Application No. PCT/US2008/009771, filed Aug. 14,2008; and U.S. Provisional Patent Application No. 60/964,715, filed Aug.14, 2007.

FIELD OF THE INVENTION

The present invention relates to specific binding members, particularlyantibodies and fragments thereof, which bind to EGFR on tumor cellsoverexpressing EGFR, and to EGFR on tumor cells that express thetruncated version of the EGFR receptor de2-7 having the in-framedeletion of exons 2 to 7 of EGFR, resulting in a truncated EGFR receptormissing 267 amino acids from the extracellular domain (de2-7 EGFR). Inparticular, the epitope recognized by the specific binding members,particularly antibodies and fragments thereof, is enhanced or evidentupon aberrant post-translational modification. These specific bindingmembers are useful in the diagnosis and treatment of cancer. The bindingmembers of the present invention may also be used in therapy incombination with chemotherapeutics or anti-cancer agents and/or withother antibodies or fragments thereof.

BACKGROUND OF RELATED TECHNOLOGY

The treatment of proliferative disease, particularly cancer, bychemotherapeutic means often relies upon exploiting differences intarget proliferating cells and other normal cells in the human or animalbody. For example, many chemical agents are designed to be taken up byrapidly replicating DNA so that the process of DNA replication and celldivision is disrupted. Another approach is to identify antigens on thesurface of tumor cells or other abnormal cells which are not normallyexpressed in developed human tissue, such as tumor antigens or embryonicantigens. Such antigens can be targeted with binding proteins such asantibodies which can block or neutralize the antigen. In addition, thebinding proteins, including antibodies and fragments thereof, maydeliver a toxic agent or other substance which is capable of directly orindirectly activating a toxic agent at the site of a tumor.

The EGFR is an attractive target for tumor-targeted antibody therapybecause it is over-expressed in many types of epithelial tumors(Voldborg et al. (1997). Epidermal growth factor receptor (EGFR) andEGFR mutations, function and possible role in clinical trials. AnnOncol. 8, 1197-206; den Eynde, B. and Scott, A. M. Tumor Antigens. In:P. J. Delves and I. M. Roitt (eds.), Encyclopedia of Immunology, SecondEdition, pp. 2424-31. London: Academic Press (1998)). Moreover,expression of the EGFR is associated with poor prognosis in a number oftumor types including stomach, colon, urinary bladder, breast, prostate,endometrium, kidney and brain (e.g., glioma). Consequently, a number ofEGFR antibodies have been reported in the literature with severalundergoing clinical evaluation (Baselga et al. (2000) Phase I Studies ofAnti-Epidermal Growth Factor Receptor Chimeric Antibody C225 Alone andin Combination With Cisplatin. J. Clin. Oncol. 18, 904; Faillot et al.(1996): A phase I study of an anti-epidermal growth factor receptormonoclonal antibody for the treatment of malignant gliomas.Neurosurgery. 39, 478-83; Seymour, L. (1999) Novel anti-cancer agents indevelopment: exciting prospects and new challenges. Cancer Treat. Rev.25, 301-12)).

Results from studies using EGFR mAbs in patients with head and neckcancer, squamous cell lung cancer, brain gliomas and malignantastrocytomas have been encouraging. The antitumor activity of most EGFRantibodies is enhanced by their ability to block ligand binding (Sturgiset al. (1994) Effects of antiepidermal growth factor receptor antibody528 on the proliferation and differentiation of head and neck cancer.Otolaryngol. Head Neck. Surg. 111, 633-43; Goldstein et al. (1995)Biological efficacy of a chimeric antibody to the epidermal growthfactor receptor in a human tumor xenograft model. Clin. Cancer Res. 1,1311-8). Such antibodies may mediate their efficacy through bothmodulation of cellular proliferation and antibody dependent immunefunctions (e.g. complement activation). The use of these antibodies,however, may be limited by uptake in organs that have high endogenouslevels of EGFR such as the liver and skin (Baselga et al., 2000; Faillotet al., 1996).

A significant proportion of tumors containing amplifications of the EGFRgene (i.e., multiple copies of the EGFR gene) also co-express atruncated version of the receptor (Wikstrand et al. (1998) The class IIIvariant of the epidermal growth factor receptor (EGFR): characterizationand utilization as an immunotherapeutic target. J. Neurovirol. 4,148-158) known as de2-7 EGFR, ΔEGFR, or A2-7 (terms used interchangeablyherein) (Olapade-Olaopa et al. (2000) Evidence for the differentialexpression of a variant EGF receptor protein in human prostate cancer.Br. J. Cancer. 82, 186-94). The rearrangement seen in the de2-7 EGFRresults in an in-frame mature mRNA lacking 801 nucleotides spanningexons 2-7 (Wong et al. (1992) Structural alterations of the epidermalgrowth factor receptor gene in human gliomas. Proc. Natl. Acad. Sci.U.S.A. 89, 2965-9; Yamazaki et al. (1990) A deletion mutation within theligand binding domain is responsible for activation of epidermal growthfactor receptor gene in human brain tumors. Jpn. J. Cancer Res. 81,773-9; Yamazaki et al. (1988) Amplification of the structurally andfunctionally altered epidermal growth factor receptor gene (c-erbB) inhuman brain tumors. Mol. Cell Biol. 8, 1816-20; Sugawa et al. (1990)Identical splicing of aberrant epidermal growth factor receptortranscripts from amplified rearranged genes in human glioblastomas.Proc. Natl. Acad. Sci. U.S.A. 87, 8602-6). The corresponding EGFRprotein has a 267 amino acid deletion comprising residues 6-273 of theextracellular domain and a novel glycine residue at the fusion junction(Sugawa et al., 1990). This deletion, together with the insertion of aglycine residue, produces a unique junctional peptide at the deletioninterface (Sugawa et al., 1990).

The de2-7 EGFR has been reported in a number of tumor types includingglioma, breast, lung, ovarian and prostate (Wikstrand et al. (1997) Cellsurface localization and density of the tumor-associated variant of theepidermal growth factor receptor, EGFRvIII. Cancer Res. 57, 4130-40;Olapade-Olaopa et al. (2000) Evidence for the differential expression ofa variant EGF receptor protein in human prostate cancer. Br. J. Cancer.82, 186-94; Wikstrand, et al. (1995) Monoclonal antibodies againstEGFRvIII in are tumor specific and react with breast and lung carcinomasand malignant gliomas. Cancer Res. 55, 3140-8; Garcia de Palazzo et al.(1993) Expression of mutated epidermal growth factor receptor bynon-small cell lung carcinomas. Cancer Res. 53, 3217-20). While thistruncated receptor does not bind ligand, it possesses low constitutiveactivity and imparts a significant growth advantage to glioma cellsgrown as tumor xenografts in nude mice (Nishikawa et al. (1994) A mutantepidermal growth factor receptor common in human glioma confers enhancedtumorigenicity. Proc. Natl. Acad. Sci. U.S.A. 91, 7727-31) and is ableto transform NIH3T3 cells (Bata et al. (1995) Epidermal growth factorligand independent, unregulated, cell-transforming potential of anaturally occurring human mutant EGFRvIII gene. Cell Growth Differ. 6,1251-9) and MCF-7 cells. The cellular mechanisms utilized by the de2-7EGFR in glioma cells are not fully defined but are reported to include adecrease in apoptosis (Nagane et al. (1996) A common mutant epidermalgrowth factor receptor confers enhanced tumorigenicity on humanglioblastoma cells by increasing proliferation and reducing apoptosis.Cancer Res. 56, 5079-86) and a small enhancement of proliferation(Nagane et al., 1996).

As expression of this truncated receptor is restricted to tumor cells itrepresents a highly specific target for antibody therapy. Accordingly, anumber of laboratories have reported the generation of both polyclonal(Humphrey et al. (1990) Anti-synthetic peptide antibody reacting at thefusion junction of deletion mutant epidermal growth factor receptors inhuman glioblastoma. Proc. Natl. Acad. Sci. U.S.A. 87, 4207-11) andmonoclonal (Wikstrand et al. (1995) Monoclonal antibodies againstEGFRvIII are tumor specific and react with breast and lung carcinomasand malignant gliomas; Okamoto et al. (1996) Monoclonal antibody againstthe fusion junction of a deletion-mutant epidermal growth factorreceptor. Br. J. Cancer. 73, 1366-72; Hills et al. (1995) Specifictargeting of a mutant, activated EGF receptor found in glioblastomausing a monoclonal antibody. Int. J. Cancer. 63, 537-43) antibodiesspecific to the unique peptide of de2-7 EGFR. A series of mouse mAbs,isolated following immunization with the unique de2-7 peptide, allshowed selectivity and specificity for the truncated receptor andtargeted de2-7 EGFR positive xenografts grown in nude mice (Wikstrand etal. (1995); Reist et al. (1997) Improved targeting of an anti-epidermalgrowth factor receptor variant III monoclonal antibody in tumorxenografts after labeling using N-succinimidyl5-iodo-3-pyridinecarboxylate. Cancer Res. 57, 1510-5; Reist et al.(1995) Tumor-specific anti-epidermal growth factor receptor variant IIImonoclonal antibodies: use of the tyramine-cellobiose radioiodinationmethod enhances cellular retention and uptake in tumor xenografts.Cancer Res. 55, 4375-82).

However, one potential shortcoming of de2-7 EGFR antibodies is that onlya proportion of tumors exhibiting amplification of the EGFR gene alsoexpress the de2-7EGFR (Ekstrand et al. (1992) Amplified and rearrangedepidermal growth factor receptor genes in human glioblastomas revealdeletions of sequences encoding portions of the N-and/or C-terminaltails. Proc. Natl. Acad. Sci. U.S.A. 89, 4309-13). The exact percentageof tumors containing the de2-7 EGFR is not completely established,because the use of different techniques (i.e. PCR versusimmunohistochemistry) and various antibodies, has produced a wide rangeof reported values for the frequency of its presence. Published dataindicates that approximately 25-30% of gliomas express de2-7 EGFR withexpression being lowest in anaplastic astrocytomas and highest inglioblastoma multiforme (Wong et al. (1992); Wikstrand et al. (1998) Theclass III variant of the epidermal growth factor receptor (EGFR):characterization and utilization as an immunotherapeutic target. J.Neurovirol. 4, 148-58; Moscatello et al. (1995) Frequent expression of amutant epidermal growth factor receptor in multiple human tumors. CancerRes. 55, 5536-9). The proportion of positive cells within de2-7 EGFRexpressing gliomas has been reported to range from 37-86% (Wikstrand etal. (1997)). 27% of breast carcinomas and 17% of lung cancers were foundto be positive for the de2-7 EGFR (Wikstrand et al. (1997); Wikstrand etal. (1995); Wikstrand et al.(1998); and Hills et al., 1995). Thus, de2-7EGFR specific antibodies would be expected to be useful in only apercentage of EGFR positive tumors.

Thus, while the extant evidence of activity of EGFR antibodies isencouraging, the observed limitations on range of applicability andefficacy reflected above remain. Accordingly, it would be desirable todevelop antibodies and like agents that demonstrate efficacy with abroad range of tumors, and it is toward the achievement of thatobjective that the present invention is directed.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention provides isolated specific binding members,particularly antibodies or fragment thereof, which recognizes an EGFRepitope which does not demonstrate any amino acid sequence alterationsor substitutions from wild-type EGFR and which is found in tumorigenic,hyperproliferative or abnormal cells and is not generally detectable innormal or wild type cells (the term “wild-type cell” as used hereincontemplates a cell that expresses endogenous EGFR but not the de2-7EGFR and the term specifically excludes a cell that overexpressesEGFR and/or the EGFR gene; the term “wild-type” refers to a genotype orphenotype or other characteristic present in a normal cell rather thanin an abnormal or tumorigenic cell).

In a further aspect, the present invention provides specific bindingmembers, particularly antibodies or fragments thereof, which recognizesan EGFR epitope which is found in tumorigenic, hyperproliferative orabnormal cells and is not generally detectable in normal or wild typecells, wherein the epitope is enhanced or evident upon aberrant posttranslational modification or aberrant expression, includingoverexpression.

In a particular non-limiting exemplification provided herein, the EGFRepitope is enhanced or evident wherein post-translational modificationis not complete or full to the extent seen with normal expression ofEGFR in wild type cells.

In one aspect, the EGFR epitope is enhanced or evident upon initial orsimple carbohydrate modification or early glycosylation, particularlyhigh mannose modification, and is reduced or not evident in the presenceof complex carbohydrate modification.

The specific binding members, which may be antibodies or fragmentsthereof, such as immunogenic fragments thereof, do not substantiallybind to or recognize normal or wild type cells containing normal or wildtype EGFR epitope in the absence of aberrant expression (includingoverexpression) and in the presence of normal EGFR post-translationalmodification.

More particularly, the specific binding member of the invention, may beantibodies or fragments thereof, which recognizes an EGFR epitope whichis present in cells overexpressing EGFR (which may, for example, resultfrom amplification of the EGFR gene) or expressing the de2-7 EGFR,particularly in the presence of aberrant post-translationalmodification, and that is not generally detectable in cells expressingEGFR under normal conditions, particularly in the presence of normalpost-translational modification.

The present inventors have discovered novel monoclonal antibodies,exemplified herein by the antibodies designated mAb806, ch806, hu806,mAb175, mAb124, and mAb1133, which specifically recognize aberrantlyexpressed (including overexpressed) EGFR.

In particular, the antibodies of the present invention recognize an EGFRepitope which is found in tumorigenic, hyperproliferative or abnormalcells and is not generally detectable in normal or wild type cells,wherein the epitope is enhanced or evident upon aberrantpost-translational modification.

The novel antibodies of the invention also recognize amplified andoverexpressed wild type EGFR and the de2-7 EGFR, yet bind to an epitopedistinct from the unique junctional peptide of the de2-7 EGFR mutation.

The antibodies of the present invention specifically recognizeaberrantly expressed EGFR, including amplified EGFR, overexpressed EGFR,and mutant EGFR (exemplified herein by the de2-7 mutation), particularlyupon aberrant post-translational modification.

Additionally, while these antibodies do not recognize the EGFR whenexpressed on the cell surface of a glioma cell line expressing normalamounts of EGFR, they do bind to the extracellular domain of the EGFR(sEGFR) immobilized on the surface of ELISA plates, indicating therecognition of a conformational epitope.

These antibodies bind to the surface of A431 cells, which have anamplification of the EGFR gene but do not express the de2-7 EGFR.Importantly, these antibodies did not bind significantly to normaltissues such as liver and skin, which express levels of endogenous, wildtype (wt) EGFR that are higher than in most other normal tissues, butwherein EGFR is not aberrantly expressed or amplified.

The antibodies of the present invention can specifically categorize thenature of EGFR tumors or tumorigenic cells, by staining or otherwiserecognizing those tumors or cells wherein aberrant EGFR expression,including EGFR amplification, EGFR overexpression and/or EGFR mutation(particularly de2-7EGFR) is present.

Further, the antibodies of the present invention demonstrate significantin vivo anti-tumor activity against tumors containingamplified/overexpressed EGFR and against de2-7 EGFR positive xenografts.

The unique specificity of these antibodies to bind to the de2-7 EGFR andamplified EGFR, but not to the normal, wild type EGFR, providesdiagnostic and therapeutic uses to identify, characterize and target anumber of tumor types, for example, head and neck, breast, or prostatetumors and glioma, without the problems associated with normal tissueuptake that may be seen with previously known EGFR antibodies.

Accordingly, the invention provides specific binding proteins, such asantibodies, which bind to the de2-7 EGFR at an epitope which is distinctfrom the junctional peptide but which do not substantially bind to EGFRon normal cells in the absence of EGFR overexpression, which may result,for example, from amplification of the EGFR gene. By amplification, itis meant, for example, to include that the cell comprises multiplecopies of the EGFR gene.

Preferably the epitope recognized by the inventive antibodies is locatedwithin the region comprising residues 273-501 of the mature normal orwild type EGFR sequence, and preferably comprises residues 287-302 (SEQID NO:14) of the mature normal or wild type EGFR sequence. Therefore,also provided are specific binding proteins, such as antibodies, whichbind to the de2-7 EGFR at an epitope located within the regioncomprising residues 273-501 and/or 287-302 (SEQ ID NO:14) of the EGFRsequence.

The epitope may be determined by any conventional epitope mappingtechniques known to the person skilled in the art. Alternatively, theDNA sequence encoding residues 273-501 and/or 287-302 (SEQ ID NO:14)could be digested, and the resultant fragments expressed in a suitablehost. Antibody binding could be determined as mentioned above.

In a preferred aspect, the antibodies are ones which have thecharacteristics of the antibodies which the inventors have identifiedand characterized, in particular recognizing aberrantly expressed EGFR,as found in amplified EGFR, overexpressed EGFR and de2-7EGFR.

In another aspect, the invention provides antibodies capable ofcompeting with the inventive antibodies, under conditions in which atleast 10% of an antibody having the VH and VL chain sequences of theinventive antibodies are blocked from binding to de2-7EGFR bycompetition with such an antibody in an ELISA assay. In particular,anti-idiotype antibodies are contemplated and are exemplified herein.The anti-idiotype antibodies LMH-11, LMH-12 and LMH-13 are providedherein.

The binding of an antibody to its target antigen is mediated through thecomplementarity-determining regions (CDRs) of its heavy and lightchains, with the role of CDR3 being of particular importance.Accordingly, specific binding members based on the CDR3 regions of theheavy or light chain, and preferably both, of the inventive antibodieswill be useful specific binding members for in vivo therapy.

Accordingly, specific binding proteins such as antibodies which arebased on the CDRs of the inventive antibodies identified, particularlythe CDR3 regions, will be useful for targeting tumors with overexpressedEGFR (including amplified EGFR) regardless of their de2-7 EGFR status.As the inventive antibodies do not bind significantly to normal, wildtype receptor, there would be no significant uptake in normal tissue, alimitation of EGFR antibodies currently being developed.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody does not bind to the de2-7 EGFR junctional peptideconsisting of the amino acid sequence of SEQ ID NO:13, wherein theantibody binds to an epitope within the sequence of residues 287-302(SEQ ID NO:14) of human wild-type EGFR, and wherein the antibody doesnot comprise a heavy chain variable region sequence having the aminoacid sequence set forth in SEQ ID NO:2 and does not comprise a lightchain variable region sequence having the amino acid sequence set forthin SEQ ID NO:4.

In another aspect, there is provided an isolated antibody wherein theantibody comprises a heavy chain and a light chain, the heavy chainhaving the amino acid sequence set forth in SEQ ID NO:42, and the lightchain having the amino acid sequence set forth in SEQ ID NO:47.

In another aspect, there is provided an isolated antibody wherein theantibody comprises a heavy chain and a light chain, the heavy chainhaving the amino acid sequence set forth in SEQ ID NO:129, and the lightchain having the amino acid sequence set forth in SEQ ID NO:134.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, the heavy chainhaving the amino acid sequence set forth in SEQ ID NO:22, and the lightchain having the amino acid sequence set forth in SEQ ID NO:27.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, the heavy chainhaving the amino acid sequence set forth in SEQ ID NO:32, and the lightchain having the amino acid sequence set forth in SEQ ID NO:37.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, wherein the variableregion of the heavy chain comprises polypeptide binding domain regionshaving amino acid sequences highly homologous to the amino acidsequences set forth in SEQ ID NOS:44, 45, and 46.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, wherein the variableregion of the light chain comprises polypeptide binding domain regionshaving amino acid sequences highly homologous to the amino acidsequences set forth in SEQ ID NOS:49, 50, and 51.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, wherein the variableregion of the heavy chain comprises polypeptide binding domain regionshaving amino acid sequences highly homologous to the amino acidsequences set forth in SEQ ID NOS:130, 131, and 132.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, wherein the variableregion of the light chain comprises polypeptide binding domain regionshaving amino acid sequences highly homologous to the amino acidsequences set forth in SEQ ID NOS:135, 136, and 137.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, wherein the variableregion of the heavy chain comprises polypeptide binding domain regionshaving amino acid sequences highly homologous to the amino acidsequences set forth in SEQ ID NOS:23, 24, and 25.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, wherein the variableregion of the light chain comprises polypeptide binding domain regionshaving amino acid sequences highly homologous to the amino acidsequences set forth in SEQ ID NOS:28, 29, and 30.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, wherein the variableregion of the heavy chain comprises polypeptide binding domain regionshaving amino acid sequences highly homologous to the amino acidsequences set forth in SEQ ID NOS:33, 34, and 35.

In another aspect, there is provided an isolated antibody, wherein theantibody comprises a heavy chain and a light chain, wherein the variableregion of the light chain comprises polypeptide binding domain regionshaving amino acid sequences highly homologous to the amino acidsequences set forth in SEQ ID NOS:38, 39, and 40.

In another aspect, there is provided an isolated antibody, wherein theisolated antibody is the form of an antibody F(ab′)2, scFv fragment,diabody, triabody or tetrabody.

In another aspect, there is provided an isolated antibody furthercomprising a detectable or functional label.

In another aspect, the detectable or functional label is a covalentlyattached drug.

In another aspect, the label is a radiolabel.

In another aspect, there is provided an isolated antibody, wherein theisolated antibody is peglyated.

In another aspect, there is provided an isolated nucleic acid whichcomprises a sequence encoding an isolated antibody recited herein.

In another aspect, there is provided a method of preparing an isolatedantibody, comprising expressing a nucleic acid as recited above andherein under conditions to bring about expression of the antibody, andrecovering the antibody.

In another aspect, there is provided a method of treatment of a tumor ina human patient which comprises administering to the patient aneffective amount of an isolated antibody recited herein.

In another aspect, there is provided a kit for the diagnosis of a tumorin which EGFR is aberrantly expressed, including overexpressed EGFR andamplified EGFR, or in which EGFR is expressed in the form of a truncatedprotein, comprising an isolated antibody recited herein.

In another aspect, the kit further comprises reagents and/orinstructions for use.

In another aspect, there is provided a pharmaceutical compositioncomprising an isolated antibody as recited herein.

In another aspect, the pharmaceutical composition further comprises apharmaceutically acceptable vehicle, carrier or diluent.

In another aspect, the pharmaceutical composition further comprises ananti-cancer agent selected from the group consisting of chemotherapeuticagents, anti-EGFR antibodies, radioimmunotherapeutic agents, chemicalablation agents, toxins, immunomodulators, cytokines, cytotoxic agents,drugs and combinations thereof.

In another aspect, the chemotherapeutic agents are selected from thegroup consisting of tyrosine kinase inhibitors, phosphorylation cascadeinhibitors, post-translational modulators, cell growth or divisioninhibitors (e.g. anti-mitotics), signal transduction inhibitors, andcombinations thereof.

In another aspect, the tyrosine kinase inhibitors are selected from thegroup consisting of AG1478, ZD1839,STI571, OSI-774, SU-6668, andcombinations thereof.

In another aspect, the anti-EGFR antibodies are selected from the groupconsisting of the anti-EGFR antibodies 528,225, SC-03,DR8. 3, L8A4, Y10,ICR62, ABX-EGF, and combinations thereof.

In another aspect, the anti-cancer agent is selected from the groupconsisting: 4-desacetylvinblastine-3-carbohydiazide;5-fluoro-2′-deoxyuridine; 5-fluorouracil; 5-fluorouracil decarbonizes;6-mercaptopurine; 6-thioguanine; abrin; abrin A chain; actinomycin D;actinomycin D, 1-dehydrotestosterone; adriamycin; alkylating agents;alkylphosphocholines; aminopterin; angiogenin; angiostatin;anthracyclines; anthramycin; anti-angiogenics; anti-folates;anti-metabolites; anti-mitotics; antibiotics; ara-C; auristatinderivatives; auristatin E; auristatin E valeryl benzylhydrazone;auristatin F phenylene diamine; auristatins; auromycins; bis-iodo-phenolmustard; bismuth; bleomycin; busulfan; calicheamicin; carboplatin;carminomycin; carmustine; cc-1065 compounds; chlorambucil;cis-dichlorodiamine platinum (cisplatin); colchicin (colchicine);combrestatin; crotin; curicin; cyclothosphamide; cytarabine;cytochalasin B; cytosine arabinoside; cytoxin; dacarbazine; dactinomycin(actinomycin); daunorubicin (daunomycin); dibromomannitol; dihydroxyanthracin dione; diphtheria toxin; dolastatin-10; doxetaxel;doxorubicin; doxorubicin hydrazides; duocarmycins; emetine; endostatin;enediyenes; enomycin; epirubicin; esperamicin compounds; ethidiumbromide; etoposide; gelonin; glucocorticoids; gramicidin D; granulocytecolony stimulating factor; granulocyte macrophage colony stimulatingfactor; idarubicin; intercalating agents; interleukin-1; interleukin-2;interleukin-6; lidocaine; lomustine; lymphokine; maytansinols;mechlorethamine; melphalan (and other related nitrogen mustards);methotrexate; minor groove-binders; mithramycin; mitogellin; mitomycinC; mitomycins; mitoxantrone; MMAF-dimethylaminoethylamine;MMAF-N-t-butyl; MMAF-tetraethylene glycol; modeccin A chain; mono-methylauristatin E (MMAE); mono-methyl auristatin F (MMAF);morpholinodoxorubicin; NT-deacetyl-N2′-(c-mercapto-1oxopropyl)-maytansine (DM1);N2′-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4);neocarzinostatin; nerve growth factor (and other growth factors);onapristone; paclitaxel; PE40; phenomycin; platelet derived growthfactor; prednisone; procaine; propranolol; Pseudomonas exotoxin A;puromycin; radioactive isotopes (such as, for example and withoutlimitation, At211, Bi212, Bi213, Cf252, I125, I131, In111, Ir192, Lu177,P32, Re186, Re188, Sm153, Y90, and W188); retstrictocin; ricin A;ricins; Sapaonaria officinalis inhibitor; saporin; streptozotocin;suramin; tamoxifen; taxanes; taxoids; taxol; tenoposide; tetracaine;thioepa chlorambucil; thiotepa; thrombotic agents; tissue plasminogenactivator; topoisomerase I inhibitors; topoisomerase II inhibitors;toxotere; tumor necrosis factor; vinblastine; vinca alkaloids; vincas;vincristine; vindesine; vinorelbine; yttrium; α-interferon; α-sarcin;and β-interferon.

In another aspect, the anti-cancer agent is conjugated to an isolatedantibody as recited herein, and may be conjugated using one or morelinker, spacer and stretcher compounds.

In another aspect, the one or more linker, space and stretcher compoundsare selected from the group consisting of: valine-citrulline;maleimidocaproyl; amino benzoic acids; p-aminobenzylcarbamoyl (PAB);lysosomal enzyme-cleavable linkers; maleimidocaproyl-polyethylene glycol(MC(PEG)6-OH); N-methyl-valine citrulline; N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB); and N-Succinimidyl4-(2-pyridylthio)pentanoate (SPP).

In another aspect, there is provided a method of preventing and/ortreating cancer in mammals, comprising administering to a mammal atherapeutically effective amount of a pharmaceutical composition asrecited herein.

In another aspect, there is provided a method for the treatment ofbrain-resident cancers that produce aberrantly expressed EGFR inmammals, comprising administering to a mammal a therapeuticallyeffective amount of a pharmaceutical composition as recited herein.

In another aspect, the brain-resident cancers are selected from thegroup consisting of glioblastomas, medulloblastomas, meningiomas,neoplastic astrocytomas and neoplastic arteriovenous malformations.

In another aspect, there is provided a unicellular host transformed witha recombinant DNA molecule which encodes an isolated antibody recitedherein.

In another aspect, the unicellular host is selected from the groupconsisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO,YB/20, NSO, SP2/0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10cells, plant cells, insect cells, and human cells in tissue culture.

In another aspect, there is provided a method for detecting the presenceof overexpressed EGFR and/or amplified EGFR, de2-7EGFR or EGFR with highmannose glycosylation wherein the EGFR is measured by: (a) contacting abiological sample from a mammal in which the presence of overexpressedEGFR and/or amplified EGFR, de2-7EGFR or EGFR with high mannoseglycosylation is suspected with an isolated antibody of claim 1 underconditions that allow binding of the EGFR to the isolated antibody tooccur; and (b) detecting whether binding has occurred between the EGFRfrom the sample and the isolated antibody; wherein the detection ofbinding indicates that presence or activity of the EGFR in the sample.

In another aspect of the method of detecting the presence ofoverexpressed EGFR and/or amplified EGFR, de2-7EGFR or EGFR with highmannose glycosylation, the detection of the presence of the EGFRindicates the existence of a tumor or cancer in the mammal.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, theheavy chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:42, and thelight chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:47.

In another aspect, the heavy chain of the antibody comprises the aminoacid sequence set forth in SEQ ID NO:42, and wherein the light chain ofthe antibody comprises the amino acid sequence set forth in SEQ IDNO:47.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, whereinthe variable region of the heavy chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:44, 45, and 46, and whereinthe variable region of the light chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:49, 50, and 51.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, theheavy chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:129, andthe light chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:134.

In another aspect, the heavy chain of the antibody comprises the aminoacid sequence set forth in SEQ ID NO:129, and wherein the light chain ofthe antibody comprises the amino acid sequence set forth in SEQ IDNO:134.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, whereinthe variable region of the heavy chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:130, 131, and 132, andwherein the variable region of the light chain comprises polypeptidebinding domain regions having amino acid sequences highly homologous tothe amino acid sequences set forth in SEQ ID NOS:135, 136, and 137.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, theheavy chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:22, and thelight chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:27.

In another aspect, the heavy chain of the antibody comprises the aminoacid sequence set forth in SEQ ID NO:22, and wherein the light chain ofthe antibody comprises the amino acid sequence set forth in SEQ IDNO:27.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, whereinthe variable region of the heavy chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:23, 24, and 25, and whereinthe variable region of the light chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:28, 29, and 30.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, theheavy chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:32, and thelight chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:37.

In another aspect, the heavy chain of the antibody comprises the aminoacid sequence set forth in SEQ ID NO:32, and wherein the light chain ofthe antibody comprises the amino acid sequence set forth in SEQ IDNO:37.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, whereinthe variable region of the heavy chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:33, 34, and 35, and whereinthe variable region of the light chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:38, 39, and 40.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody does not bind to the de2-7 EGFR junctional peptideconsisting of the amino acid sequence of SEQ ID NO:13, wherein theantibody binds to an epitope within the sequence of residues 287-302 ofhuman wild-type EGFR,

the antibody comprising a light chain and a heavy chain, wherein thevariable region of the light chain comprises a first polypeptide bindingdomain region having an amino acid sequence corresponding to the aminoacid sequence set forth in Formula I:

HSSQDIXaa₁SNIG   (I),

wherein Xaa_(i) is an amino acid residue having an uncharged polar Rgroup (SEQ ID NO:151);

a second polypeptide binding domain region having an amino acid sequencecorresponding to the amino acid sequence set forth in Formula II:

HGTNLXaa₂D   (II),

wherein Xaa₂ is an amino acid residue having a charged polar R group(SEQ ID NO:152);

and a third polypeptide binding domain region having an amino acidsequence corresponding to the amino acid sequence set forth in FormulaIII:

VQYXaa₃QFPWT   (III),

wherein Xaa₃ is selected from the group consisting of A, G, and an aminoacid residue which is conservatively substituted for A or G (SEQ IDNO:153); and

wherein the variable region of the heavy chain comprises a firstpolypeptide binding domain region having an amino acid sequencecorresponding to the amino acid sequence set forth in Formula IV:

SDXaa₄AWN   (IV),

wherein Xaa₄ is selected from the group consisting of F, Y, and an aminoacid residue which is conservatively substituted for F or Y (SEQ IDNO:154);

a second polypeptide binding domain region having an amino acid sequencecorresponding to the amino acid sequence set forth in Formula V, FormulaVI, or Formula VII:

YISYSGNTRYXaa₅PSLKS   (V),

wherein Xaa₅ is an amino acid residue having an uncharged polar R group(SEQ ID NO:155),

YISYSXaa₆NTRYNPSLKS   (VI),

wherein Xaa₆ is selected from the group consisting of G, A, and an aminoacid residue which is conservatively substituted for G or A (SEQ IDNO:156),

YISYSGNTRYNPSLXaa₇S   (VII),

and Xaa ₇ is a basic amino acid residue (SEQ ID NO:157); and

a third polypeptide binding domain region having an amino acid sequencecorresponding to the amino acid sequence set forth in Formula VIII:

Xaa₈TAGRGFPY   (VIII),

wherein Xaa₈ is selected from the group consisting of V, A, and an aminoacid residue which is conservatively substituted for V or A (SEQ IDNO:158),

and wherein the antibody does not comprise a heavy chain variable regionsequence having the amino acid sequence set forth in SEQ ID NO:2 anddoes not comprise a light chain variable region sequence having theamino acid sequence set forth in SEQ ID NO:4.

In another aspect, X_(aa1) is N; X_(aa2) is D; X_(aa3) is A; X_(aa4) isF; X_(aa5) is an amino acid residue having an uncharged polar R group;X_(aa6) is G; X_(aa 7) is K; and X_(aa8) is V.

In another aspect, X_(aa5) is N or Q.

In another aspect, X_(aa1) is N or S.

In another aspect, X_(aa2) is D or E.

In another aspect, X_(aa3) is A or G.

In another aspect, X_(aa4) is F or Y.

In another aspect, X_(aa5) is N or Q.

In another aspect, X_(aa6) is G or A, and X_(aa 7) is independently K orR.

In another aspect, X_(aa8) is V or A.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody does not bind to the de2-7 EGFR junctional peptideconsisting of the amino acid sequence of SEQ ID NO:13, wherein theantibody binds to an epitope within the sequence of residues 273-501 ofhuman wild-type EGFR,

the antibody comprising a light chain and a heavy chain, wherein thevariable region of the light chain comprises a first polypeptide bindingdomain region having the amino acid sequence HSSQDINSNIG (SEQ ID NO:18);a second polypeptide binding domain region having the amino acidsequence HGTNLDD (SEQ ID NO:19); and a third polypeptide binding domainregion having the amino acid sequence VQYAQFPWT (SEQ ID NO:20),

wherein the variable region of the heavy chain comprises a firstpolypeptide binding domain region having the amino acid sequence SDFAWN(SEQ ID NO:15); a second polypeptide binding domain region having anamino acid sequence corresponding to the amino acid sequence set forthin Formula IX:

YISYSGNTRYX_(aa9)PSLKS   (IX),

wherein X_(aa9) is an amino acid residue having an uncharged polar Rgroup (SEQ ID NO:159); and

a third polypeptide binding domain region having the amino acid sequenceVTAGRGFPY (SEQ ID NO:17).

In another aspect, the antibody binds to an epitope within the sequenceof residues 287-302 (SEQ ID NO:14) of human wild-type EGFR.

In another aspect, X_(aa9) is N or Q.

In another aspect, the binding domain regions are carried by a humanantibody framework.

In another aspect, the human antibody framework is a human IgG1 antibodyframework.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, theheavy chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:2, and thelight chain having an amino acid sequence that is substantiallyhomologous to the amino acid sequence set forth in SEQ ID NO:4.

In another aspect, the heavy chain of the antibody comprises the aminoacid sequence set forth in SEQ ID NO:2, and wherein the light chain ofthe antibody comprises the amino acid sequence set forth in SEQ ID NO:4.

In another aspect, there is provided an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody comprises a heavy chain and a light chain, whereinthe variable region of the heavy chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:15, 16, and 17, and whereinthe variable region of the light chain comprises polypeptide bindingdomain regions having amino acid sequences highly homologous to theamino acid sequences set forth in SEQ ID NOS:18, 19, and 20.

In another aspect, there is provided a pharmaceutical compositioncomprising: (1) a first therapeutically active agent, comprising anisolated antibody capable of binding EGFR on tumor cells thatoverexpress EGFR, and on tumor cells that express the truncated versionof the EGFR receptor, de2-7 EGFR, wherein the antibody does not bind tothe de2-7 EGFR junctional peptide consisting of the amino acid sequenceof SEQ ID NO:13, wherein the antibody binds to an epitope within thesequence of residues 287-302 of human wild-type EGFR; and (2) a secondtherapeutically active agent.

In another aspect, the pharmaceutical composition includes an isolatedantibody comprising a heavy chain and light chain, wherein the variableregion of the heavy chain comprises polypeptide binding domain regionscorresponding to amino acids 26-36, 50-65, and 97-105 of SEQ ID NO: 11,and wherein the variable region of the light chain comprises polypeptidebinding domain regions corresponding to amino acids 24-34, 50-56, and89-97 of SEQ ID NO: 12.

In another aspect, the pharmaceutical composition includes an isolatedantibody selected from the group consisting of: (1) an isolated antibodycomprising a heavy chain and a light chain, wherein the variable regionof the heavy chain comprises polypeptide binding domain regions havingthe amino acid sequences set forth in SEQ ID NOS:23, 24, and 25, andwherein the variable region of the light chain comprises polypeptidebinding domain regions having amino acid sequences set forth in SEQ IDNOS:28, 29, and 30; (2) an isolated antibody comprising a heavy chainand a light chain, wherein the variable region of the heavy chaincomprises polypeptide binding domain regions having the amino acidsequences set forth in SEQ ID NOS:33, 34, and 35, and wherein thevariable region of the light chain comprises polypeptide binding domainregions having amino acid sequences set forth in SEQ ID NOS:38, 39, and40; and (3) an isolated antibody comprising a heavy chain and a lightchain, wherein the variable region of the heavy chain comprisespolypeptide binding domain regions having the amino acid sequences setforth in SEQ ID NOS:130, 131, and 132, and wherein the variable regionof the light chain comprises polypeptide binding domain regions havingamino acid sequences set forth in SEQ ID NOS:135, 136, and 137.

In another aspect, the pharmaceutical composition includes an isolatedantibody comprising a heavy chain and a light chain, wherein thevariable region of the heavy chain comprises polypeptide binding domainregions having the amino acid sequences set forth in SEQ ID NOS:44, 45,and 46, and wherein the variable region of the light chain comprisespolypeptide binding domain regions having amino acid sequences set forthin SEQ ID NOS:49, 50, and 51.

In another aspect, the second therapeutically active agent of thepharmaceutical composition is an anti-cancer agent, which in certainaspects may be selected from the group consisting of erlotinib,5-fluorouracil, cisplatin, a combination of 5-fluorouracil andcisplatin, bevacizumab, and cetuximab.

In another aspect, the second therapeutically active agent of thepharmaceutical composition is an anti-cancer agent, which in certainaspects is a tyrosine kinase inhibitor, which in certain aspects may beselected from the group consisting of AG1478, ZD1839,STI571, OSI-774,SU-6668, and combinations thereof.

In another aspect, the second therapeutically active agent of thepharmaceutical composition is an anti-cancer agent, which in certainaspects is anti-EGFR antibody, which in certain aspects may be selectedfrom the group consisting of the anti-EGFR antibodies 528, SC-03,DR8. 3,L8A4, Y10, ICR62, ABX-EGF, and combinations thereof.

In another aspect, the second therapeutically active agent of thepharmaceutical composition is an anti-cancer agent, which in certainaspects may be selected from the group consisting of4-desacetylvinblastine-3-carbohydiazide; 5-fluoro-2′-deoxyuridine;5-fluorouracil decarbonizes; 6-mercaptopurine; 6-thioguanine; abrin;abrin A chain; actinomycin D; actinomycin D, 1-dehydrotestosterone;adriamycin; alkylating agents; alkylphosphocholines; aminopterin;angiogenin; angiostatin; anthracyclines; anthramycin; anti-angiogenics;anti-folates; anti-metabolites; anti-mitotics; antibiotics; ara-C;auristatin derivatives; auristatin E; auristatin E valerylbenzylhydrazone; auristatin F phenylene diamine; auristatins;auromycins; bis-iodo-phenol mustard; bismuth; bleomycin; busulfan;calicheamicin; carboplatin; carminomycin; carmustine; cc-1065 compounds;chlorambucil; colchicin (colchicine); combrestatin; crotin; curicin;cyclothosphamide; cytarabine; cytochalasin B; cytosine arabinoside;cytoxin; dacarbazine; dactinomycin (actinomycin); daunorubicin(daunomycin); dibromomannitol; dihydroxy anthracin dione; diphtheriatoxin; dolastatin-10; doxetaxel; doxorubicin; doxorubicin hydrazides;duocarmycins; emetine; endostatin; enediyenes; enomycin; epirubicin;esperamicin compounds; ethidium bromide; etoposide; gelonin;glucocorticoids; gramicidin D; granulocyte colony stimulating factor;granulocyte macrophage colony stimulating factor; idarubicin;intercalating agents; interleukin-1; interleukin-2; interleukin-6;lidocaine; lomustine; lymphokine; maytansinols; mechlorethamine;melphalan (and other related nitrogen mustards); methotrexate; minorgroove-binders; mithramycin; mitogellin; mitomycin C; mitomycins;mitoxantrone; MMAF-dimethylaminoethylamine; MMAF-N-t-butyl;MMAF-tetraethylene glycol; modeccin A chain; mono-methyl auristatin E(MMAE); mono-methyl auristatin F (MMAF); morpholinodoxorubicin;NT-deacetyl-N2′-(c-mercapto-1 oxopropyl)-maytansine (DM1);N2′-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4);neocarzinostatin; nerve growth factor (and other growth factors);onapristone; paclitaxel; PE40; phenomycin; platelet derived growthfactor; prednisone; procaine; propranolol; Pseudomonas exotoxin A;puromycin; radioactive isotopes (such as, for example and withoutlimitation, At211, Bi212, Bi213, Cf252, I125, I131, In111, Ir192, Lu177,P32, Re186, Re188, Sm153, Y90, and W188); retstrictocin; ricin A;ricins; Sapaonaria officinalis inhibitor; saporin; streptozotocin;suramin; tamoxifen; taxanes; taxoids; taxol; tenoposide; tetracaine;thioepa chlorambucil; thiotepa; thrombotic agents; tissue plasminogenactivator; topoisomerase I inhibitors; topoisomerase II inhibitors;toxotere; tumor necrosis factor; vinblastine; vinca alkaloids; vincas;vincristine; vindesine; vinorelbine; yttrium; α-interferon; α-sarcin;and β-interferon.

In another aspect, there is provided a method for the treatment ofcancer in mammals, comprising administering to a mammal atherapeutically effective amount of (1) an isolated antibody capable ofbinding EGFR on tumor cells that overexpress EGFR, and on tumor cellsthat express the truncated version of the EGFR receptor, de2-7 EGFR,wherein the antibody does not bind to the de2-7 EGFR junctional peptideconsisting of the amino acid sequence of SEQ ID NO:13, wherein theantibody binds to an epitope within the sequence of residues 287-302 ofhuman wild-type EGFR; and (2) one or more doses of radiation.

In another aspect, there is provided a method for the treatment ofcancer in mammals, comprising administering to a mammal atherapeutically effective amount of a pharmaceutical compositioncomprising: (1) a first therapeutically active agent, comprising anisolated antibody capable of binding EGFR on tumor cells thatoverexpress EGFR, and on tumor cells that express the truncated versionof the EGFR receptor, de2-7 EGFR, wherein the antibody does not bind tothe de2-7 EGFR junctional peptide consisting of the amino acid sequenceof SEQ ID NO:13, wherein the antibody binds to an epitope within thesequence of residues 287-302 of human wild-type EGFR; and (2) a secondtherapeutically active agent.

In another aspect, the pharmaceutical composition administered intreating cancer in a mammal includes an isolated antibody selected fromthe group consisting of: (1) an isolated antibody comprising a heavychain and light chain, wherein the variable region of the heavy chaincomprises polypeptide binding domain regions corresponding to aminoacids 26-36, 50-65, and 97-105 of SEQ ID NO: 11, and wherein thevariable region of the light chain comprises polypeptide binding domainregions corresponding to amino acids 24-34, 50-56, and 89-97 of SEQ IDNO: 12; (2) an isolated antibody comprising a heavy chain and a lightchain, wherein the variable region of the heavy chain comprisespolypeptide binding domain regions having the amino acid sequences setforth in SEQ ID NOS:23, 24, and 25, and wherein the variable region ofthe light chain comprises polypeptide binding domain regions havingamino acid sequences set forth in SEQ ID NOS:28, 29, and 30; (3) anisolated antibody comprising a heavy chain and a light chain, whereinthe variable region of the heavy chain comprises polypeptide bindingdomain regions having the amino acid sequences set forth in SEQ IDNOS:33, 34, and 35, and wherein the variable region of the light chaincomprises polypeptide binding domain regions having amino acid sequencesset forth in SEQ ID NOS:38, 39, and 40; and (4) an isolated antibodycomprising a heavy chain and a light chain, wherein the variable regionof the heavy chain comprises polypeptide binding domain regions havingthe amino acid sequences set forth in SEQ ID NOS:130, 131, and 132, andwherein the variable region of the light chain comprises polypeptidebinding domain regions having amino acid sequences set forth in SEQ IDNOS:135, 136, and 137.

In another aspect, the pharmaceutical composition administered intreating cancer in a mammal includes a second therapeutically activeagent which is an anti-cancer agent, which in certain aspects may beselected from the group consisting of erlotinib, 5-fluorouracil,cisplatin, a combination of 5-fluorouracil and cisplatin, bevacizumab,and cetuximab.

In another aspect, the cancer that is treated in a mammal is abrain-resident cancer that produces aberrantly expressed EGFR, which maybe selected from the group consisting of glioblastomas,medulloblastomas, meningiomas, neoplastic astrocytomas and neoplasticarteriovenous malformations.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing detailed description, whichproceeds with reference to the following illustrative drawings, and theattendant claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the results of flow cytometric analysis of glioma celllines. U87MG (light gray histograms) and U87MG.Δ2-7 (dark grayhistograms) cells were stained with either an irrelevant IgG2b antibody(open histograms), DH8.3 (specific for de2-7 EGFR), mAb806 or 528 (bindsboth wild type and de2-7 EGFR) as indicated.

FIGS. 2A-D present the results of ELISA of mAb806, mAbDH8.3 and mAb528.(A) binding of increasing concentrations of mAb806 (▴) DH8.3 () or 528(▪) antibody to sEGFR coated ELISA plates. (B) inhibition of mAb806 andmAb528 binding to sEGFR coated ELISA plates by increasing concentrationsof soluble EGFR (sEGFR) in solution. (C) binding of increasingconcentrations of DH8.3 to the de2-7 junctional peptide illustratesbinding curves for mAb806 and mAb528 to immobilized wild-type sEGFR (D).

FIGS. 2E and 2F graphically present the results of BIAcore bindingstudies using C-terminal biotinylated peptide and including a monoclonalantibody of the invention, along with other known antibodies, among themthe L8A4 antibody which recognizes the junction peptide of the de2-7EGFR mutant, and controls.

FIG. 3 depicts the internalization of mAb806 and the DH8.3 antibody.U87MG.Δ2-7 cells were pre-incubated with mAb806 (▴) or DH8.3 (•) at 4°C., transferred to 37° C. and internalization determined by FACS. Datarepresents mean internalization at each time point ±SE of 3 (DH8.3) or 4(mAb806) separate experiments.

FIGS. 4A and 4B illustrate biodistribution (% ID/g tumor tissue) ofradiolabeled (a) ¹²⁵I-mAb806 and (b) ¹³¹I-DH8.3 in nude mice bearingU87MG and U87MG.Δ2-7 xenografts. Each point represents the mean of 5mice±SE except for 1 hr where n=4.

FIGS. 5A and 5B illustrate biodistribution of radiolabeled ¹²⁵I-mAb806(open bar) and ¹³¹I-DH8.3 (filled bar) antibodies expressed as (a)tumor:blood or (b) tumor:liver ratios in nude mice bearing U87MG.Δ2-7xenografts. Each bar represents the mean of 5 mice±SE except for 1 hrwhere n=4

FIGS. 6A-C illustrate flow cytometric analysis of cell lines containingamplification of the EGFR gene. A431 cells were stained with eithermAb806, DH8.3 or 528 (black histograms) and compared to an irrelevantIgG2b antibody (open histogram).

FIGS. 7A and 7B illustrate biodistribution (% ID/g tumor tissue) ofradiolabeled (a) ¹²⁵I-mAb806 and (b) ¹³¹I-528 in nude mice bearingU87MG.Δ2-7 and A431 xenografts.

FIGS. 8A-D illustrate biodistribution of radiolabeled ¹²⁵I-mAb806 (openbar) and ¹³¹I-528 (filled bar) and antibodies expressed as (A, B)tumor:blood or (C, D) tumor:liver ratios in nude mice bearing (A, C)U87MG.Δ2-7 and (B, D) A431 xenografts.

FIGS. 9A and 9B illustrate anti-tumor effect of mAb806 on (A) U87MG and(B) U87MG.Δ2-7 xenograft growth rates in a preventative model. 3×10⁶U87MG or U87MG.Δ2-7 cells were injected s.c. into both flanks of 4-6week old BALB/c nude mice, (n=5) at day 0. Mice were injected i.p. witheither 1 mg of mAb806 (•); 0.1 mg of mAb806 (▴); or vehicle (∘) startingone day prior to tumor cell inoculation. Injections were given threetimes per week for two weeks as indicated by the arrows. Data areexpressed as mean tumor volume±S.E.

FIGS. 10A, 10B, and 10C illustrate the anti-tumor effect of mAb806 on(A) U87MG, (B) U87MG.Δ2-7 and (C) U87MG.wtEGFR xenografts in anestablished model. 3×10⁶ U87MG, U87MG.Δ2-7, or U87MG.wtEGFR cells, wereinjected s.c. into both flanks of 4-6 week old BALB/c nude mice, (n=5).Mice were injected i.p. with either 1 mg doses of mAb806 (•); 0.1 mgdoses of mAb806 (▴); or vehicle (∘) starting when tumors had reached amean tumor volume of 65-80 mm³. Injections were given three times perweek for two weeks as indicated by the arrows. Data are expressed asmean tumor volume±S.E.

FIGS. 11A and 11B illustrate anti-tumor effect of mAb806 on A431xenografts in (A) preventative and (B) established models. 3×10⁶ A431cells were injected s.c. into both flanks of 4-6 week old BALB/c nudemice (n=5). Mice were injected i.p. with either 1 mg doses of mAb806(•); or vehicle (∘), starting one day prior to tumor cell inoculation inthe preventative model, or when tumors had reached a mean tumor volumeof 200 mm³. Injections were given three times per week for two weeks asindicated by the arrows. Data are expressed as mean tumor volume±S.E.

FIG. 12 illustrates the anti-tumor effect of treatment with mAb806combined with treatment with AG1478 on A431 xenografts in a preventativemodel. Data are expressed as mean tumor volume±S.E.

FIG. 13 depicts mAb806 binding to A431 cells in the presence ofincreasing concentrations of AG 1478 (0.5 μM and 5 μM).

FIGS. 14A and 14B illustrate the (A) nucleic acid sequence and the (B)amino acid translation thereof of the 806 VH chain gene (SEQ ID NO:1 andSEQ ID NO:2, respectively).

FIGS. 15A and 15B illustrate the (A) nucleic acid sequence and the (B)amino acid translation thereof of the 806 VL chain gene (SEQ ID NO:3 andSEQ ID NO:4, respectively).

FIG. 16 shows the VH chain sequence (SEQ ID NO:2) numbered according toKabat, with the CDRs (SEQ ID NOS:15, 16 and 17) underlined. Key residuesof the VH chain sequence (SEQ ID NO:2) are 24, 37, 48, 67 and 78.

FIG. 17 shows the VL chain sequence (SEQ ID NO:4) numbered according toKabat, with the CDRs (SEQ ID NOS:18, 19 and 20) underlined. Key residuesof the VL chain sequence (SEQ ID NO:4) are 36, 46, 57 and 71.

FIGS. 18A-18D show the results of in vivo studies designed to determinethe therapeutic effect of combination antibody therapy, particularlymAb806 and the 528 antibody. Mice received inoculations of U87MG.D2-7 (Aand B),U87MG.DK (C), or A431 (D) cells.

FIGS. 19A-D show analysis of internalization by electron microscopy.U87MG.Δ2-7 cells were pre-incubated with mAb806 or DH8.3 followed bygold conjugated anti-mouse IgG at 4° C., transferred to 37° C. andinternalization examined at various time points by electron microscopy.(A) localization of the DH8.3 antibody to a coated pit (arrow) after 5min; (B) internalization of mAb806 by macropinocytosis (arrow) after 2min; (C) localization of DH8.3 to lysosomes (arrow) after 20 min; (D)localization of mAb806 to lysosomes (arrow) after 30 min. Originalmagnification for all images is ×30,000.

FIG. 20 shows autoradiography of a U87MG.Δ2-7 xenograft sectioncollected 8 hr after injection of ¹²⁵I-mAb806.

FIG. 21 shows flow cytometric analysis of cell lines containingamplification of the EGFR gene. HN5 and MDA-468 cells were stained withan irrelevant IgG2b antibody (open histogram with dashed line), mAb806(black histogram) or 528 (open histogram with closed lines). The DH8.3antibody was completely negative on both cell lines (data not shown).

FIG. 22 shows immunoprecipitation of EGFR from cell lines. The EGFR wasimmunoprecipitated from ³⁵S-labeled U87MG.Δ2-7 or A431 cells withmAb806, sc-03 antibody or a IgG2b isotype control. Arrows at the sideindicate the position of the de2-7 and wt EGFR. Identical bandingpatterns were obtained in 3 independent experiments.

FIG. 23 shows autoradiography of an A431 xenograft section collected 24hr after injection of ¹²⁵I-mAb806, areas of localization to viabletissue are indicated (arrows).

FIGS. 24A and 24B show extended survival of nude mice bearingintracranial U87MG.ΔEGFR (A) and LN-Z308.ΔEGFR (B) xenografts withsystemic mAb806 treatment. U87MG.EGFR cells (1×10⁵) or LN-Z308.ΔEGFRcells (5×10⁵) were implanted into nude mice brains, and the animals weretreated with either mAb806, PBS, or isotype IgG from post-implantationdays 0 through 14.

FIGS. 24C and 24D show growth inhibition of intracranial tumors bymAb806 treatment. Nude mice (five per group), treated with either mAb806or the isotype IgG control, were euthanized on day 9 for U87MG.EGFR (C)and on day 15 for LN-Z308.ΔEGFR (D), and their brains were harvested,fixed, and sectioned. Data were calculated by taking the tumor volume ofcontrol as 100%. Values are mean±SD. * * * , P<0.001; control versusmAb806. Arrowheads, tumor tissue.

FIG. 24E shows extended survival of nude mice bearing intracranialU87MG.ΔEGFR xenografts with intratumoral mAb806 treatment. U87MG.ΔEGFRcells were implanted as described. 10 mg of mAb806 or isotype IgGcontrol in a volume of 5 μl were injected at the tumor-injection siteevery other day starting at day 1 for five times.

FIGS. 25A, 25B, and 25C show that mAb806 extends survival of mice withU87MG.wtEGFR brain tumors but not with U87MG.DK. or U87MG brain tumors.U87MG (A), U87MG.DK (B), or U87MG.wtEGFR (C) cells (5×10⁵) wereimplanted into nude mice brains, and the animals were treated withmAb806 from post-implantation days 0 through 14 followed by observationafter discontinuation of therapy.

FIG. 26A shows FACS analysis of mAb806 reactivity with U87MG cell lines.U87MG, U87MG.ΔEGFR, U87MG.DK, and U87MG.wtEGFR cells were stained withanti-EGFR mAbs 528, EGFR.1, and anti-ΔEGFR antibody, mAb806. MonoclonalEGFR. 1 antibody recognized wtEGFR exclusively and monoclonal 528antibody reacted with both wtEGFR and ΔEGFR. mAb806 reacted intensivelywith U87MG.ΔEGFR and U87MG.DK and weakly with U87MG.wtEGFR. Bars on theabscissa, maximum staining of cells in the absence of primary antibody.Results were reproduced in three independent experiments.

FIG. 26B shows mAb806 immunoprecipitation of EGFR forms. Mutant andwtEGFR were immunoisolated with anti-EGFR antibodies, 528, EGFR. 1, oranti-ΔEGFR antibody, mAb806, from (Lane 1) U87MG, (Lane 2) U87Δ.EGFR,(Lane 3) U87MG.DK, and (Lane 4) U87MG.wtEGFR cells, and were thendetected by Western blotting with anti-pan EGFR antibody, C13.

FIGS. 27A and 27B show that systemic treatment with mAb806 decreases thephosphorylation of ΔEGFR and Bcl-XL expression in U87MG.ΔEGFR braintumors. U87MG.ΔEGFR tumors were resected at day 9 of mAb806 treatment,immediately frozen in liquid nitrogen and stored at −80° C. before tumorlysate preparation.

(A) Western blot analysis of expression and the degree ofautophosphorylation of ΔEGFR. Thirty μg of tumor lysates were subjectedto SDS-polyacrylamide gels, transferred to nitrocellulose membranes, andprobed with anti-phosphotyrosine mAb, then were stripped and re-probedwith anti-EGFR antibody, C13.

(B) Western blotting of Bcl-XL by using the same tumor lysates as in(A). Membranes were probed with anti-human Bcl-X polyclonal antibody.Lanes 1 and 2, U87MG.ΔEGFR brain tumors treated with isotype control;Lanes 3 and 4, U87MG.ΔEGFR brain tumors treated with mAb806.

FIG. 28 shows mAb806 treatment leads to a decrease in growth andvasculogenesis and to increases in apoptosis and accumulatingmacrophages in U87MG.ΔEGFR tumors. Tumor sections were stained forKi-67. Cell proliferative index was assessed by the percentage of totalcells that were Ki-67 positive from four randomly selected high powerfields (×400) in intracranial tumors from four mice of each group. Dataare the mean±SE. Apoptotic cells were detected by TUNEL assay. Apoptoticindex was assessed by the ratio of TUNEL-positive cells: total number ofcells from four randomly selected high-power fields (×400) inintracranial tumors from four mice of each group. Data are the mean±SE.Tumor sections were immunostained with anti-CD31 antibody. MVAs wereanalyzed by computerized image analysis from four randomly selectedfields (×200) from intracranial tumors from four mice of each group.Peritumoral infiltrates of macrophages in mAb806-treated U87MG.ΔEGFRtumors. Tumor sections were stained with anti-F4/80 antibody.

FIG. 29 shows flow cytometric analysis of parental and transfected U87MGglioma cell lines. Cells were stained with either an irrelevant IgG2bantibody (open histograms) or the 528 antibody or mAb806 (filledhistograms) as indicated.

FIG. 30 shows immunoprecipitation of EGFR from cell lines. The EGFR wasimmunoprecipitated from ³⁵S-labeled U87MG.wtEGFR, U87MG.Δ2-7, and A431cells with mAb806 (806), sc-03 antibody (c-term), or a IgG2b isotypecontrol (con). Arrows, position of the de2-7 and wt EGFR.

FIG. 31 shows representative H&E-stained paraffin sections of U87MG.Δ2-7and U87MG.wtEGFR xenografts. U87MG.Δ2-7 (collected 24 days after tumorinoculation) and U87MG.wtEGFR (collected 42 days after tumorinoculation) xenografts were excised from mice treated as described inFIG. 10 above, and stained with H&E. Vehicle-treated U87MG.Δ2-7(collected 18 days after tumor inoculation) and U87MG.wtEGFR (collected37 days after tumor inoculation) xenografts showed very few areas ofnecrosis (left panel), whereas extensive necrosis (arrows) was observedin both U87MG.Δ2-7 and U87MG.wtEGFR xenografts treated with mAb806(right panel).

FIG. 32 shows immunohistochemical analysis of EGFR expression in frozensections derived from U87MG, U87MG.Δ2-7, and U87MG.wtEGFR xenografts.Sections were collected at the time points described in FIG. 31 above.Xenograft sections were immunostained with the 528 antibody (left panel)and mAb806 (right panel). No decreased immunoreactivity to eitherwtEGFR, amplified EGFR, or de2-7 EGFR was observed in xenografts treatedwith mAb806. Consistent with the in vitro data, parental U87MGxenografts were positive for 528 antibody but were negative for mAb806staining.

FIG. 33 shows a schematic representation of generated bicistronicexpression constructs. Transcription of the chimeric antibody chains isinitiated by Elongation Factor-1 promoter and terminated by a strongartificial termination sequence. IRES sequences were introduced betweencoding regions of light chain and NeoR and heavy chain and dhfr gene.

FIGS. 34A and 34B show biodistribution analysis of the ch806radiolabeled with either (A) ¹²⁵I or (B) ¹¹¹In was performed in BALB/cnude mice bearing U87MG-de2-7 xenograft tumors. Mice were injected with5 μg of radiolabeled antibody and in groups of 4 mice per time point,sacrificed at either 8, 28, 48 or 74 hours. Organs were collected,weighed and radioactivity measured in a gamma counter.

FIGS. 35A and 35B depict (A) the % ID gram tumor tissue and (B) thetumor to blood ratio. Indium-111 antibody shows approximately 30%ID/gram tissue and a tumor to blood ratio of 4.0.

FIG. 36 depicts the therapeutic efficacy of chimeric antibody ch806 inan established tumor model. 3×10⁶ U87MG.Δ2-7 cells in 100 μl of PBS wereinoculated s.c. into both flanks of 4-6 week old female nude mice.mAb806 was included as a positive control. Treatment was started whentumors had reached a mean volume of 50 mm³ and consisted of 1 mg ofch806 or mAb806 given i.p. for a total of 5 injections on the daysindicated. Data was expressed as mean tumor volume±S.E. for eachtreatment group.

FIG. 37 shows CDC Activity on Target (A) U87MG.de2-7 and (B) A431 cellsfor anti-EGFR chimeric IgGI antibodies ch806 and control cG250. Mean(bars; ±SD) percent cytotoxicity of triplicate determinations arepresented.

FIG. 38 shows ADCC on target (A) U87MG.de2-7 and (B) A431 cells atEffector:Target cell ratio of 50:1 mediated by ch806 and isotype controlcG250 (0-10 μg/ml). Results are expressed as mean (bars; ±SD) percentcytotoxicity of triplicate determinations.

FIG. 39 shows ADCC mediated by 1 μg/ml parental mAb806 and ch806 ontarget U87MG.de2-7 cells over a range of Effector:Target ratios. Mean(bars; ±SD) of triplicate determinations are presented.

FIG. 40 shows twenty-five hybridomas producing antibodies that boundch806 but not huIgG were initially selected. Four of these anti-ch806hybridomas with high affinity binding (clones 3E3, 5B8, 9D6 and 4D8)were subsequently pursued for clonal expansion from single cells bylimiting dilution and designated Ludwig Institute for Cancer ResearchMelbourne Hybridoma (LMH)-11, -12, -13 and -14, respectively. Inaddition, two hybridomas that produced mAbs specific for huIgG were alsocloned and characterized further: clones 2C10 (LMH-15) and 2B8 (LMH-16).

FIGS. 41A, 41B, and 41C show that after clonal expansion, the hybridomaculture supernatants were examined in triplicate by ELISA for theability to neutralize ch806 or mAb806 antigen binding activity withsEGFR621. Mean (±SD) results demonstrated the antagonist activity ofanti-idiotype mAbs LMH-11, -12, -13 and -14 with the blocking insolution of both ch806 and murine mAb806 binding to plates coated withsEGFR (LMH-14 not shown).

FIGS. 42A, 42B, and 42C show microtitre plates that were coated with 10μg/ml purified (A) LMH-11, (B) LMH-12 and (C) LMH-13. The three purifiedclones were compared for their ability to capture ch806 or mAb806 insera or 1% FCS/Media and then detect bound ch806 or mAb806. Isotypecontrol antibodies hu3S193 and m3S193 in serum and 1% FCS/Media wereincluded in addition to controls for secondary conjugate avidin-HRP andABTS substrate. Results are presented as mean (±SD) of triplicatesamples using biotinylated-LMH-12 (10 μg/ml) for detection and indicateLMH-12 used for capture and detection had the highest sensitivity forch806 in serum (3 ng/ml) with negligible background binding.

FIG. 43 shows validation of the optimal pharmacokinetic ELISA conditionsusing 1 μg/ml anti-idiotype LMH-12 and 1 μg/mlbiotinylated LMH-12 forcapture and detection, respectively. Three separate ELISAs wereperformed in quadruplicate to measure ch806 in donor serum () fromthree healthy donors or 1% BSA/media (▪) with isotype control hu3S193 inserum (▴) or 1% BSA/media (▾). Controls for secondary conjugateavidin-HRP (♦) and ABTS substrate (hexagon) alone were also includedwith each ELISA. Mean (±SD) results demonstrate highly reproduciblebinding curves for measuring ch806 (2 μg/ml-1.6 ng/ml) in sera with a 3ng/ml limit of detection. (n=12; 1-100 ng/ml, Coefficient of Variation<25%; 100 ng/ml-5 μg/ml, Coefficient of Variation <15%). No backgroundbinding was evident with any of the three sera tested and negligiblebinding was observed with isotype control hu3S193.

FIG. 44 depicts an immunoblot of recombinant sEGFR expressed in CHOcells, blotted with mAb806. Recombinant sEGFR was treated with PNGaseFto remove N-linked glycosylation (deglycosylated), or untreated(untreated), the protein was run on SDS-PAGE, transferred to membraneand immunoblotted with mAb806.

FIG. 45 depicts immunoprecipitation of EGFR from ³⁵S-labelled cell lines(U87MG.Δ2-7, U87MG-wtEGFR, and A431) with different antibodies (SC-03,806 and 528 antibodies).

FIG. 46 depicts immunoprecipitation of EGFR from different cells (A431and U87MG.Δ2-7) at different time points (time 0 to 240 minutes) afterpulse-labeling with ³⁵S methionine/cysteine. Antibodies 528 and 806 areused for immunoprecipitation.

FIG. 47 depicts immunoprecipitation of EGFR from various cell lines(U87MGΔ2-7, U87MG-wtEGFR and A431) with various antibodies (SC-03, 806and 528) in the absence of (−) and after Endo H digestion (+) to removehigh mannose type carbohydrates.

FIG. 48 depicts cell surface iodination of the A431 and U87MG.Δ2-7 celllines followed by immunoprecipitation with the 806 antibody, and with orwithout Endo H digestion, confirming that the EGFR bound by mAb806 onthe cell surface of A431 cells is an EndoH sensitive form.

FIG. 49 shows the pREN ch806 LC Neo Vector (SEQ ID NO:7).

FIG. 50 shows the pREN ch806 HC DHFR Vector (SEQ ID NO:8).

FIGS. 51A-D shows the mAb124 VH and VL chain nucleic acid sequences (SEQID NOS:21 and 26, respectively) and amino acid sequences (SEQ ID NOS:22and 27, respectively).

FIGS. 52A-D shows the mAb1133 VH and VL chain nucleic acid sequences(SEQ ID NO:31 and 36, respectively) and amino acid sequences (SEQ IDNOS:32 and 37, respectively).

FIG. 53 shows a DNA plasmid graphic of the combined, double gene Lonzaplasmid including pEE12.4 containing the hu806H (VH+CH) expressioncartridge, and pEE6.4 containing the hu806L (VL+CL) expressioncartridge.

FIG. 54 shows the DNA sequence (SEQ ID NO:41; complement SEQ ID NO:162)of the combined Lonza plasmid described in FIG. 53. This sequence alsoshows all translations (SEQ ID NOS:42-51 and 163-166) relevant to thehu806 antibody. The plasmid has been sequence-verified, and the codingsequence and translation checked. Sections of the sequence have beenshaded to identify regions of interest; the shaded regions correspond toactual splice junctions. The color code is as follows:

(gray): signal region, initial coding sequences found at both the heavyand light-chain variable regions;

(lavender): hu806 VH chain, veneered heavy-chain variable region;

(pink): hu806 CH chain , codon-optimized heavy-chain constant region;

(green): hu806 VL chain, veneered light-chain variable region; and

(yellow): hu806 CL chain, codon-optimized light-chain constant region.

FIGS. 55A and 55B show the hu806 translated amino acid sequences (VH andVL chains of SEQ ID NOS:164 and 166 and their respective signal peptidesof SEQ ID NOS:163 and 165; CH and CL chains of SEQ ID NOS:43 and 48),and give the Kabat numbers for the VH and VL chains (SEQ ID NOS:164 and165, respectively), with CDRs (SEQ ID NOS:44-46 and 49-51) underlined.

FIGS. 56A, 56B, 56C, 57A, 57B, and 57C show the initial step inveneering design, the grading of amino acid residues in the mAb806sequence (VH chain of SEQ ID NO:167 and VL chain of SEQ ID NO:12) forsurface exposure. Grades are given in the number of asterisks (*) aboveeach residue, with the most exposed residues having three asterisks.These figures include a design indicating how the initialoligonucleotides (VH chain: FIG. 56C and SEQ ID NOS:52 and 169-177; VLchain: FIG. 57C and SEQ ID NOS: 62, 66, 68 and 181-187) overlapped toform the first veneered product (VH chain of SEQ ID NO:168 and VL chainof SEQ ID NO:180).

FIG. 58 shows a map of codon optimized huIgG1 heavy chain DNA sequence(SEQ ID NO:80; complement SEQ ID NO:178) and amino acid translation (SEQID NO:43).

FIG. 59 shows the protein alignment comparing the hu806 VH+CH amino acidsequence (8C65AAG hu806 VH+CH; SEQ ID NO:81) to the original referencefile for the mAb806 VH chain (SEQ ID NO:167). Highlighted regionsindicate conserved amino acid sequences in the VH chain. The CDRs areunderlined. Asterisks reflect changes that were planned and carried outin the initial veneering process. The numbered sites are references tolater modifications.

FIG. 60 shows the corresponding alignment for the hu806 VL+CL amino acidsequence (8C65AAG hu806 signal+VL+CL; SEQ ID NO:83) to the originalreference file for the mAb806 VL chain (SEQ ID NO:179). It contains anadditional file (r2vk1 hu806 signal+VL+CL; SEQ ID NO:82), a precursorconstruct, which was included to illustrate the change made atmodification #7.

FIG. 61 shows a nucleotide and amino acid alignment of the hu806signal+VL and CL sequences (8C65AAG hu806 V1+Cl; SEQ ID NOS:190 and 188)with the corresponding ch806 sequences (pREN ch806 LC Neo; LICR; SEQ IDNO:189). It has been modified and annotated as described in FIG. 62.

FIG. 62 shows the nucleotide alignment of the hu806 signal+VH sequence(8C65AAG hu806 VH chain; SEQ ID NO:192) with the corresponding mAb806sequence [mAb806 VH chain before codon change (cc) and veneering (yen);SEQ ID NO:191]. The nucleotide changes behind the amino acid changes ofFIGS. 59 and 60 are illustrated, as well as showing conservative nucleicacid changes that led to no change in amino acid. The intron between thesignal and the VH chain in hu806 has been removed for easier viewing.The signal sequence and CDRs are underlined. The corresponding aminoacid sequence (SEQ ID NO:42) has been superimposed on the alignment.

FIG. 63. shows binding of purified hu806 antibody obtained fromtransient transfectant 293 cells to recombinant EGFR-ECD as determinedby Biacore. No binding to the EGFR-ECD was observed with purifiedcontrol human IgG1 antibody.

FIG. 64 shows the GenBank formatted text document of the sequence (SEQID NO:41) and annotations of plasmid 8C65AAG encoding the IgG1 hu806.

FIG. 65 shows the alignment of amino acid sequences for CDRs from mAb806(SEQ ID NOS:15-18, 20 and 193) and mAb175 (SEQ ID NOS:130-132, 135 and194-195). Sequence differences between the two antibodies are bolded.

FIGS. 66A and 66B show immunohistochemical staining of cell lines andnormal human liver with mAb175. (A) Biotinylated mAb175 was used tostain sections prepared from blocks containing A431 cells (over-expressthe wtEGFR), U87MG.Δ2-7 cells (express the Δ2-7EGFR) and U87MG cells(express the wtEGFR at modest levels). (B) Staining of normal humanliver (400×) with mAb175 (left panel), isotype control (centre panel)and secondary antibody control (right panel). No specific sinusoidal orhepatocyte staining was observed.

FIGS. 67A, 67B, and 67C show the reactivity of mAb806 and mAb175 withfragments of the EGFR displayed on yeast. (A) Representative flowcytometry histograms depicting the mean fluorescence signal of mAb175and mAb806-labeling of yeast-displayed EGFR fragments. With yeastdisplay a percentage of cells do not express protein on their surfaceresulting in 2 histogram peaks. The 9E10 antibody is used as a positivecontrol as all fragments contain a linear C-terminal c-myc tag. (B)Summary of antibody binding to various EGFR fragments. (C) The EGFRfragments were denatured by heating yeast pellets to 800° C. for 30 min.The c-myc tag was still recognized by the 9E10 anti-myc antibody in allcases, demonstrating that heat treatment does not compromise the yeastsurface displayed protein. The conformation sensitive EGFR antibodymAb225 was used to confirm denaturation.

FIGS. 68A, 68B, 68C, and 68D show the antitumor effects of mAb175 onbrain and prostate cancer xenografts. (A) Mice (n=5) bearing U87MG.Δ2-7xenografts were injected i.p. with PBS, 1 mg of mAb175 or mAb806(positive control), three times weekly for two weeks on days 6, 8, 10,13, 15 and 17 when the starting tumor volume was 100 mm³. Data areexpressed as mean tumor volume±SE. (B) Cells were stained with twoirrelevant antibodies (blue, solid and green, hollow), mAb 528 for totalEGFR (pink, solid), mAb806 (light blue, hollow) and mAb175 (orange,hollow) and then analyzed by FACS. (C) DU145 cells were lysed, subjectedto IP with mAb 528, mAb806, mAb175 or two independent irrelevantantibodies and then immunoblotted for EGFR. (D) Mice (n=5) bearing DU145xenografts were injected i.p. with PBS, 1 mg of mAb175 or mAb806, dailyon days 18-22, 25-29 and 39-43 when the starting tumor volume was 85mm³. Data are expressed as mean tumor volume±SE.

FIGS. 69A, 69B, 69C, 69D, 69E, and 69F show the crystal structures ofEGFR peptide 287-302 bound to the Fab fragments (A) Cartoon of Fab 806,with the light chain, red; heavy chain, blue; bound peptide, yellow; andthe superposed EGFR₂₈₇₋₃₀₂ from EGFR, purple. (B) Cartoon of Fab 175with the light chain, yellow; heavy chain, green; bound peptide, lilac;and EGFR₂₈₇₋₃₀₂ from EGFR(DI-3), purple. (C) Detail from (B) showing thesimilarity of EGFR₂₈₇₋₃₀₂ in the receptor to the peptide bound to FAb175. Peptides backbones are shown as Ca traces and the interacting sidechains as sticks. O atoms are colored red; N, blue; S, orange and C, asfor the main chain. (D) Superposition of EGFR with the Fab175:peptidecomplex showing spacial overlap. Coloring as in (C) with the surface ofEGFR187-286 colored turquoise. (E) Orthogonal view to (D) withEGFR187-286 shown in opaque blue and the surface of the light (orange)and heavy (green) chains transparent. (F) Detailed stereoview of 175 Fabcomplex looking into the antigen-binding site. Coloring as in (C) andside chain hydrogen bonds dotted in black. Water molecules buried uponcomplex formation are shown as red spheres.

FIGS. 70A, 70B, 70C, and 70D show the influence of the 271-283 cysteinebond on mAb806 binding to the EGFR. (A) Cells transfected with wtEGFR,EGFR-C271A, EGFR-C283A or the C271A/C283A mutant were stained withmAb528 (solid pink histogram), mAb806 (blue line) or only the secondaryantibody (purple) and then analyzed by FACS. The gain was set up using aclass-matched irrelevant antibody. (B) BaF3 cells expressing theEGFR-C271A or C271/283A EGFR were examined for their response to EGF inan MTT assay as described. EC₅₀₅ were derived using the Bolzman fit ofthe data points. Data represent mean and sd of triplicate measurements.(C) BaF3 cells expressing the wild-type or the EGFR-C271A/C283A wereIL-3 and serum starved, then exposed to EGF or vehicle control. Wholecell lysates were separated by SDS-PAGE and immunoblotted withanti-phosphotyrosine antibody (top panel) or anti-EGFR antibody (bottompanel). (D) BaF3 cells expressing the wild-type (left panel) or theC271A/C283A (right panel) EGFR were stimulated with increasingconcentrations of EGF in the presence of no antibody (open symbols), mAb528 (grey circles) or mAb806 (black triangles), both at 10 μg/ml. Dataare expressed as mean and sd of triplicate measurements.

FIGS. 71A, 71B, and 71C show: (A) Whole body gamma camera image of thebiodistribution of ¹¹¹In ch806 in a patient with metastatic squamouscell carcinoma of the vocal cord, showing quantitative high uptake intumor in the right neck (arrow). Blood pool activity, and minorcatabolism of free ¹¹¹In in liver, is also seen. (B) Single PhotonComputed Tomography (SPECT) image of the neck of this patient, showinguptake of ¹¹¹In-ch806 in viable tumor (arrow), with reduced centraluptake indicating necrosis. (C) Corresponding CT scan of the neckdemonstrating a large right neck tumor mass (arrow) with centralnecrosis.

FIGS. 72A and 72B show a stereo model of the structure of the untetheredEGFR1-621. The receptor backbone is traced in blue and the ligand TGF-αin red. The mAb806/175 epitope is drawn in turquoise and the disulfidebonds in yellow. The atoms of the disulfide bond which ties the epitopeback into the receptor are shown in space-filling format. The model wasconstructed by docking the EGFR-ECD CR2 domain from the tetheredconformation onto the structure of an untethered EGFR monomer in thepresence of its ligand.

FIG. 73 shows the reactivity of mAb806 with fragments of the EGFR.Lysates from 293T cells transfected with vectors expressing the soluble1-501 EGFR fragment or GH/EGFR fragment fusion proteins (GH-274-501,GH-282-501, GH-290-501 and GH-298-501) were resolved by SDS-PAGE,transferred to membrane and immunoblotted with mAb806 (left panel) orthe anti-myc antibody 9B11 (right panel).

FIGS. 74A and 74B show the mAb175 VH chain nucleic acid sequence (SEQ IDNO:128) and amino acid sequence (SEQ ID NO:129), respectively.

FIGS. 75A and 75B show the mAb175 VL chain nucleic acid sequence (SEQ IDNO:133) and amino acid sequence (SEQ ID NO:134), respectively.

FIGS. 76A, 76B, and 76C show: (A) Volumetric product concentration and(B) viable cell concentration of GS-CHO (14D8, 15B2 and 40A10) andGS-NS0 (36) hu806 transfectants in small scale (100 mL) shake flaskscultures. Product concentration was estimated by ELISA using the 806anti-idiotype as coating antibody and ch806 Clinical Lot: J06024 asstandard; (C) GS-CHO 40A10 transfectant cell growth and volumetricproduction in a 15 L stirred tank bioreactor. Viable cell density (♦×10⁵cell/mL), cell viability (▪) and production (^(▴)mg/L).

FIGS. 77A, 77B, 77C, 77D, and 77E show Size Exclusion Chromatography(Biosep SEC-S3000) Analysis of Protein-A purified hu806 antibodyconstructs produced by small scale culture and control ch806 and mAb806. Chromatograms at A214 nm are presented in the upper panels and atA280 nm in the lower panel of each Figure.

FIG. 78 shows Size Exclusion Chromatography (Biosep SEC-S3000) Analysisof Protein-A purified hu806 antibody construct 40A10 following largescale production and Protein-A purification. Chromatogram at A214 nm ispresented indicating 98.8% purity with 1.2% aggregate present.

FIG. 79 shows that precast 4-20% Tris/Glycine Gels from Novex, USA wereused under standard SDS-PAGE conditions to analyze purified transfectanthu806 preparations (5 μg) GS CHO (14D8, 15B2 and 40A10) and GS-NS0 (36)hu806 under reduced conditions. Proteins detected by Coomassie BlueStain.

FIG. 80 shows that precast 4-20% Tris/Glycine Gels were used understandard SDS-PAGE conditions to analyze purified transfectant hu806preparations (5 μg) GS CHO (14D8, 15B2 and 40A10) and GS-NS0 (36) undernon-reduced conditions. Proteins detected by Coomassie Blue Stain.

FIG. 81 shows that precast 4-20% Tris/Glycine Gels were used understandard SDS-PAGE conditions to analyze purified transfectant hu806 GSCHO 40A10 (5 μg) following large scale production. Proteins detected byCoomassie Blue Stain.

FIG. 82 shows Isoelectric Focusing gel analysis of purified transfectanthu806 GS CHO 40A10 (5 μg) following 15 L production. Proteins detectedby Coomassie Blue Stain. Lane 1, pI markers; Lane 2, hu806 (threeisoforms, pI 8.66 to 8.82); Lane 3, pI markers.

FIG. 83 shows binding to A431 cells: Flow Cytometry analysis ofProtein-A purified hu806 antibody preparations (20 μg/ml), and isotypecontrol huA33 (20 μg/ml). Controls include secondary antibody alone(green) and ch806 (red). Hu806 constructs were produced by small scaleculture.

FIG. 84 shows binding to A431 cells: Flow Cytometry analysis of purifiedmAb806, ch806 and hu806 40A10 antibody preparations (20 μg/ml) that bind˜10% of wild type EGFR on cell surface, 528 (binds both wild type andde2-7 EGFR) and irrelevant control antibody (20 μg/ml) as indicated.

FIG. 85 shows binding to U87MG.de2-7 glioma cells. Flow Cytometryanalysis of purified mAb806, ch806 and hu806 40A10 antibody preparations(20 μg/ml) and 528 anti-EGFR and irrelevant control antibody (20 μg/ml).

FIG. 86 shows specific binding of ¹²⁵I-radiolabelled 806 antibodyconstructs to: (A) U87MG.de2-7 glioma cells and (B) A431 carcinomacells.

FIG. 87 shows Scatchard Analyses: ¹²⁵I-radiolabelled (A) ch806 and (B)hu806 antibody constructs binding to U87MG.de2-7 cells.

FIG. 88 shows Scatchard Analyses: ¹²⁵I-radiolabelled (A) ch806 and (B)hu806 antibody constructs binding to A431 cells.

FIG. 89 shows BIAcore analysis of binding to 287-302 EGFR 806 peptideepitope by (A) hu806 and (B) ch806 passing over the immobilized peptidein increasing concentrations of 50 nM, 100 nM, 150 nM, 200 nM, 250 nMand 300 nM.

FIG. 90 shows ch806- and hu806-mediated Antibody Dependant CellularCytotoxicity on target A431 cells determined at (A) 1 μg/ml eachantibody over a range of effector to target cell ratios (E:T=0.78:1 to100:1); (B) at E:T=50:1 over a concentration range of each antibody(3.15 ng/ml-10 μg/ml).a on target A431.

FIG. 91 shows treatment of established A431 xenografts in BALB/c nudemice. Groups of 5 mice received 6×1 mg dose over 2 weeks antibodytherapy as indicated (arrows). Mean±SEM tumor volume is presented untilstudy termination.

FIG. 92 shows treatment of established U87MG.de2-7 xenografts in BALB/cnude mice. Groups of 5 mice received 6×1 mg dose over 2 weeks antibodytherapy as indicated (arrows). Mean±SEM tumor volume is presented untilstudy termination.

FIG. 93 shows deviations from random coil chemical shift values for themAb806 peptide (A) N, (B) HN and (C) HA. Peptide was prepared in H₂Osolution containing 5% ₂H₂O, 70 mM NaCl and 50 mM NaPO₄ at pH 6.8. Allspectra used for sequential assignments were acquired at 298K on aBruker Avance500.

FIGS. 94A, 94B, 94C, 94D, 94E, and 94F show whole body gamma cameraimages of Patient 7 A) Anterior, and B) Posterior, Day 5 post infusionof ¹¹¹In-ch806. High uptake of ¹¹¹In-ch806 in metastatic lesions in thelungs (arrows) is evident. C) and D) show metastatic lesions (arrows) onCT scan. E) 3D SPECT images of the chest, and F) co-registeredtransaxial images of SPECT and CT showing specific uptake of ¹¹¹In-ch806in metastatic lesions.

FIGS. 95A, 95B, 95C, 95D, 95E, and 95F show planar images of the headand neck of Patient 8 obtained A) Day 0, B) Day 3 and C) Day 7 postinfusion of ¹¹¹In-ch806. Initial blood pool activity is seen on Day 0,and uptake of ¹¹¹In-ch806 in an anaplastic astrocytoma in the rightfrontal lobe is evident by Day 3 (arrow), and increases by Day 7.Specific uptake of ¹¹¹In-ch806 is confirmed in D) SPECT image of thebrain (arrow), at the site of tumor (arrow) evident in E) ¹⁸F-FDG PET,and F) MRI.

FIGS. 96A, 96B, 96C, and 96D show similar uptake of 111In-ch806 in tumoris evident in Patient 3 compared to Patient 4, despite differences in806 antigen expression in screened tumor samples. A) ¹¹¹In-ch806localization in lung metastasis (arrow) on SPECT transaxial image inPatient 4, with cardiac blood pool activity (B) evident. B)corresponding CT scan. Archived tumor was shown to have <10% positivityfor 806 expression. C) ¹¹¹In-ch806 localization in lung metastasis(arrow) in Patient 3, with cardiac blood pool activity (B) evident. D)corresponding CT scan. Archived tumor was shown to have 50-75%positivity for 806 expression.

FIG. 97 shows pooled population pharmacokinetics of ch806 proteinmeasured by ELISA. Observed and predicted ch806 (% ID/L) vs. time postinfusion (hrs).

FIGS. 98A and 98B show individual patient results for A) NormalisedWhole Body Clearance and B) Hepatic Clearance of ¹¹¹In-ch806 at the 5mg/m² (▪), 10 mg/m² (Δ), 20 mg/m² (∇), and 40 mg/m² (♦) dose levels.Linear regression for data sets indicated in each panel [A) r²=0.9595;B) r²=0.9415].

FIG. 99 illustrates the effect of combination hu806 and radiationtreatment on tumor growth in xenograft models.

FIG. 100 illustrates the effect of combination hu806 and bevacizumabtreatment on tumor growth in xenograft models.

FIG. 101 illustrates the effect of combination hu806 and cetuximabtreatment on tumor growth in xenograft models.

FIG. 102 illustrates the effect of combination hu806 and erlotinibtreatment on tumor growth in xenograft models.

FIG. 103 illustrates the effect of combination hu806 and 5-fluorouraciltreatment on tumor growth in xenograft models.

FIG. 104 illustrates the effect of combination hu806 and cisplatintreatment on tumor growth in xenograft models.

FIG. 105 illustrates the effect of combination hu806, 5-fluorouracil,and cisplatin treatment on tumor growth in xenograft models.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, for example, Sambrook et al., “MolecularCloning: A Laboratory Manual” (1989); “Current Protocols in MolecularBiology” Volumes I-E [Ausubel, R. M., ed. (1994)]; “Cell Biology: ALaboratory Handbook” Volumes I-III [J. E. Celis, ed. (1994))]; “CurrentProtocols in Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

As used herein, the following terms are deemed to have, withoutlimitation, the provided definitions.

The term “specific binding member” describes a member of a pair ofmolecules which have binding specificity for one another. The members ofa specific binding pair may be naturally derived or wholly or partiallysynthetically produced. One member of the pair of molecules has an areaon its surface, or a cavity, which specifically binds to and istherefore complementary to a particular spatial and polar organizationof the other member of the pair of molecules. Thus the members of thepair have the property of binding specifically to each other. Examplesof types of specific binding pairs are antigen-antibody, biotin-avidin,hormone-hormone receptor, receptor-ligand, enzyme-substrate. Thisapplication is concerned with antigen-antibody type reactions.

The term “aberrant expression” in its various grammatical forms may meanand include any heightened or altered expression or overexpression of aprotein in a tissue, e.g. an increase in the amount of a protein, causedby any means including enhanced expression or translation, modulation ofthe promoter or a regulator of the protein, amplification of a gene fora protein, or enhanced half-life or stability, such that more of theprotein exists or can be detected at any one time, in contrast to anonoverexpressed state. Aberrant expression includes and contemplatesany scenario or alteration wherein the protein expression orpost-translational modification machinery in a cell is taxed orotherwise disrupted due to enhanced expression or increased levels oramounts of a protein, including wherein an altered protein, as inmutated protein or variant due to sequence alteration, deletion orinsertion, or altered folding is expressed.

It is important to appreciate that the term “aberrant expression” hasbeen specifically chosen herein to encompass the state where abnormal(usually increased) quantities/levels of the protein are present,irrespective of the efficient cause of that abnormal quantity or level.Thus, abnormal quantities of protein may result from overexpression ofthe protein in the absence of gene amplification, which is the case e.g.in many cellular/tissue samples taken from the head and neck of subjectswith cancer, while other samples exhibit abnormal protein levelsattributable to gene amplification.

In this latter connection, certain of the work of the inventors that ispresented herein to illustrate the invention includes the analysis ofsamples certain of which exhibit abnormal protein levels resulting fromamplification of EGFR. This therefore accounts for the presentationherein of experimental findings where reference is made to amplificationand for the use of the terms “amplification/amplified” and the like indescribing abnormal levels of EGFR. However, it is the observation ofabnormal quantities or levels of the protein that defines theenvironment or circumstance where clinical intervention as by resort tothe binding members of the invention is contemplated, and for thisreason, the present specification considers that the term “aberrantexpression” more broadly captures the causal environment that yields thecorresponding abnormality in EGFR levels.

Accordingly, while the terms “overexpression” and “amplification” intheir various grammatical forms are understood to have distincttechnical meanings, they are to be considered equivalent to each other,insofar as they represent the state where abnormal EGFR protein levelsare present in the context of the present invention. Consequently, theterm “aberrant expression” has been chosen as it is believed to subsumethe terms “overexpression” and “amplification” within its scope for thepurposes herein, so that all terms may be considered equivalent to eachother as used herein.

The term “antibody” describes an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anypolypeptide or protein having a binding domain which is, or ishomologous to, an antibody binding domain. CDR grafted antibodies arealso contemplated by this term.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding member or substancehaving a binding domain with the required specificity. Thus, this termcovers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, including any polypeptide comprising animmunoglobulin binding domain, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an immunoglobulin bindingdomain, or equivalent, fused to another polypeptide are thereforeincluded. Cloning and expression of chimeric antibodies are described inEP-A-0120694 and EP-A-0125023 and U.S. Pat. Nos. 4,816,397 and4,816,567.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al. (1989) Nature 341,544-546) which consistsof a VH domain; (v) isolated CDR regions; (vi) F (ab′) 2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al. (1988) Science. 242,423-426; Huston etal. (1988) PNAS USA. 85,5879-5883); (viii) multivalent antibodyfragments (scFv dimers, trimers and/or tetramers (Power and Hudson(2000) J. Immunol. Methods 242, 193-204) (ix) bispecific single chain Fvdimers (PCT/US92/09965) and (x) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (W094/13804; P.Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90,6444-6448).

An “antibody combining site” is that structural portion of an antibodymolecule comprised of light chain or heavy and light chain variable andhypervariable regions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F (ab′) Z and F (v), whichportions are preferred for use in the therapeutic methods describedherein.

Antibodies may also be bispecific, wherein one binding domain of theantibody is a specific binding member of the invention, and the otherbinding domain has a different specificity, e.g. to recruit an effectorfunction or the like. Bispecific antibodies of the present inventioninclude wherein one binding domain of the antibody is a specific bindingmember of the present invention, including a fragment thereof, and theother binding domain is a distinct antibody or fragment thereof,including that of a distinct anti-EGFR antibody, for instance antibody528 (U.S. Pat. No. 4,943,533), the chimeric and humanized 225 antibody(U.S. Pat. No. 4,943,533 and WO/9640210), an anti-de2-7 antibody such asDH8.3 (Hills, D. et al (1995) Int. J. Cancer. 63(4), 537-543), antibodyL8A4 and Y10 (Reist, C J et al. (1995) Cancer Res. 55 (19):4375-4382;Foulon C F et al. (2000) Cancer Res. 60 (16):44534460), ICR62(Modjtahedi H et al. (1993) Cell Biophys. January-June; 22 (1-3):129-46;Modjtahedi et al. (2002) P. A. A. C. R. 55 (14):3140-3148, or theantibody of Wikstrand et al (Wikstrand C. et al (1995) Cancer Res. 55(14):3140-3148). The other binding domain may be an antibody thatrecognizes or targets a particular cell type, as in a neural or glialcell-specific antibody. In the bispecific antibodies of the presentinvention the one binding domain of the antibody of the invention may becombined with other binding domains or molecules which recognizeparticular cell receptors and/or modulate cells in a particular fashion,as for instance an immune modulator (e.g., interleukin (s)), a growthmodulator or cytokine (e.g. tumor necrosis factor (TNF), andparticularly, the TNF bispecific modality demonstrated in U.S. Ser. No.60/355,838 filed Feb. 13, 2002, incorporated herein in its entirety) ora toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor.

Fab and F(ab′)₂ portions of antibody molecules may be prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See, for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced from F(ab′) 2 portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may alsocontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term “antigen binding domain” describes the part of an antibodywhich comprises the area which specifically binds to and iscomplementary to part or all of an antigen. Where an antigen is large,an antibody may bind to a particular part of the antigen only, whichpart is termed an epitope. An antigen binding domain may be provided byone or more antibody variable domains. Preferably, an antigen bindingdomain comprises an antibody light chain variable region (VL) and anantibody heavy chain variable region (VH).

“Post-translational modification” may encompass any one of orcombination of modification(s), including covalent modification, which aprotein undergoes after translation is complete and after being releasedfrom the ribosome or on the nascent polypeptide co-translationally.Post-translational modification includes but is not limited tophosphorylation, myristylation, ubiquitination, glycosylation, coenzymeattachment, methylation and acetylation. Post-translational modificationcan modulate or influence the activity of a protein, its intracellularor extracellular destination, its stability or half-life, and/or itsrecognition by ligands, receptors or other proteins. Post-translationalmodification can occur in cell organelles, in the nucleus or cytoplasmor extracellularly.

The term “specific” may be used to refer to the situation in which onemember of a specific binding pair will not show any significant bindingto molecules other than its specific binding partner(s). The term isalso applicable where e.g. an antigen binding domain is specific for aparticular epitope which is carried by a number of antigens, in whichcase the specific binding member carrying the antigen binding domainwill be able to bind to the various antigens carrying the epitope.

The term “comprise” generally used in the sense of include, that is tosay permitting the presence of one or more features or components.

The term “consisting essentially of” refers to a product, particularly apeptide sequence, of a defined number of residues which is notcovalently attached to a larger product. In the case of the peptide ofthe invention referred to above, those of skill in the art willappreciate that minor modifications to the N- or C-terminal of thepeptide may however be contemplated, such as the chemical modificationof the terminal to add a protecting group or the like, e.g. theamidation of the C-terminus.

The term “isolated” refers to the state in which specific bindingmembers of the invention, or nucleic acid encoding such binding memberswill be, in accordance with the present invention. Members and nucleicacid will be free or substantially free of material with which they arenaturally associated such as other polypeptides or nucleic acids withwhich they are found in their natural environment, or the environment inwhich they are prepared (e.g. cell culture) when such preparation is byrecombinant DNA technology practiced in vitro or in vivo. Members andnucleic acid may be formulated with diluents or adjuvants and still forpractical purposes be isolated—for example the members will normally bemixed with gelatin or other carriers if used to coat microtitre platesfor use in immunoassays, or will be mixed with pharmaceuticallyacceptable carriers or diluents when used in diagnosis or therapy.Specific binding members may be glycosylated, either naturally or bysystems of heterologous eukaryotic cells, or they may be (for example ifproduced by expression in a prokaryotic cell) unglycosylated.

Also, as used herein, the terms “glycosylation” and “glycosylated”includes and encompasses the post-translational modification ofproteins, termed glycoproteins, by addition of oligosaccarides.Oligosaccharides are added at glycosylation sites in glycoproteins,particularly including N-linked oligosaccharides and 0-linkedoligosaccharides. N-linked oligosaccharides are added to an Asn residue,particularly wherein the Asn residue is in the sequence N-X-S/T, where Xcannot be Pro or Asp, and are the most common ones found inglycoproteins. In the biosynthesis of N-linked glycoproteins, a highmannose type oligosaccharide (generally comprised of dolichol,N-Acetylglucosamine, mannose and glucose is first formed in theendoplasmic reticulum (ER). The high mannose type glycoproteins are thentransported from the ER to the Golgi, where further processing andmodification of the oligosaccharides occurs. 0-linked oligosaccharidesare added to the hydroxyl group of Ser or Thr residues. In 0-linkedoligosaccharides, N-Acetylglucosamine is first transferred to the Ser orThr residue by N-Acetylglucosaminyltransferase in the ER. The proteinthen moves to the Golgi where further modification and chain elongationoccurs. O-linked modifications can occur with the simple addition of theOGlcNAc monosaccharide alone at those Ser or Thr sites which can alsounder different conditions be phosphorylated rather than glycosylated.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

The terms “806 antibody”, “mAb806”, “ch806”, and any variants notspecifically listed, may be used herein interchangeably, and as usedthroughout the present application and claims refer to proteinaceousmaterial including single or multiple proteins, and extends to thoseproteins having the amino acid sequence data described herein andpresented in SEQ ID NO:2 and SEQ ID NO:4, and the chimeric antibodych806 which is incorporated in and forms a part of SEQ ID NOS:7 and 8,and the profile of activities set forth herein and in the Claims.Accordingly, proteins displaying substantially equivalent or alteredactivity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “806 antibody”, “mAb806” and “ch806” areintended to include within their scope proteins specifically recitedherein as well as all substantially homologous analogs and allelicvariations.

The terms “humanized 806 antibody”, “hu806”, and “veneered 806 antibody”and any variants not specifically listed, may be used hereininterchangeably, and as used throughout the present application andclaims refer to proteinaceous material including single or multipleproteins, and extends to those proteins having the amino acid sequencedata described herein and presented in SEQ ID NO:42 and SEQ ID NO:47,and the profile of activities set forth herein and in the Claims.Accordingly, proteins displaying substantially equivalent or alteredactivity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “humanized 806 antibody”, “hu806”, and“veneered 806 antibody” are intended to include within their scopeproteins specifically recited herein as well as all substantiallyhomologous analogs and allelic variations.

The terms “175 antibody” and “mAb175”, and any variants not specificallylisted, may be used herein interchangeably, and as used throughout thepresent application and claims refer to proteinaceous material includingsingle or multiple proteins, and extends to those proteins having theamino acid sequence data described herein and presented in SEQ ID NO:129and SEQ ID NO:134, and the profile of activities set forth herein and inthe Claims. Accordingly, proteins displaying substantially equivalent oraltered activity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “175 antibody” and “mAb175” are intendedto include within their scope proteins specifically recited herein aswell as all substantially homologous analogs and allelic variations.

The terms “124 antibody” and “mAb124”, and any variants not specificallylisted, may be used herein interchangeably, and as used throughout thepresent application and claims refer to proteinaceous material includingsingle or multiple proteins, and extends to those proteins having theamino acid sequence data described herein and presented in SEQ ID NO:22and SEQ ID NO:27, and the profile of activities set forth herein and inthe Claims. Accordingly, proteins displaying substantially equivalent oraltered activity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “124 antibody” and “mAb124” are intendedto include within their scope proteins specifically recited herein aswell as all substantially homologous analogs and allelic variations.

The terms “1133 antibody” and “mAb1133”, and any variants notspecifically listed, may be used herein interchangeably, and as usedthroughout the present application and claims refer to proteinaceousmaterial including single or multiple proteins, and extends to thoseproteins having the amino acid sequence data described herein andpresented in SEQ ID NO:32 and SEQ ID NO:37, and the profile ofactivities set forth herein and in the Claims. Accordingly, proteinsdisplaying substantially equivalent or altered activity are likewisecontemplated. These modifications may be deliberate, for example, suchas modifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in hosts that areproducers of the complex or its named subunits. Also, the terms “11133antibody” and “mAb1133” are intended to include within their scopeproteins specifically recited herein as well as all substantiallyhomologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

Table of Correspondence Symbol 1-Letter 3-Letter Amino Acid Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn aspargine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the non-transcribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream(3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′end of the primer, with theremainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding specific binding members(antibodies) of the invention which code for antibodies having thedisclosed sequences but which are degenerate to such sequences. By“degenerate to” is meant that a different three-letter codon is used tospecify a particular amino acid. It is well known in the art that thefollowing codons can be used interchangeably to code for each specificamino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (He or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Valor V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in, for example, the disclosed sequences ofantibodies of the present invention, such that a particular codon ischanged to a codon which codes for a different amino acid. Such amutation is generally made by making the fewest nucleotide changespossible. A substitution mutation of this sort can be made to change anamino acid in the resulting protein in a non-conservative manner (i.e.,by changing the codon from an amino acid belonging to a grouping ofamino acids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingprotein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

-   Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine,    Tryptophan, Methionine    Amino Acids with Uncharged Polar R Groups-   Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine,    Glutamine    Amino Acids with Charged Polar R Groups (Negatively Charged at ph    6.0)-   Aspartic acid, Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0)

-   Lysine, Arginine, Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

-   Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i. e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

-   Lys for Arg and vice versa such that a positive charge may be    maintained;-   Glu for Asp and vice versa such that a negative charge may be    maintained;-   Ser for Thr such that a free —OH can be maintained; and-   Gin for Asn such that a free NH2 can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces. (3-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, preferably by at least 50 percent, preferably by at least 70percent, preferably by at least 80 percent, preferably by at least 90%,a clinically significant change in the growth or progression or mitoticactivity of a target cellular mass, group of cancer cells or tumor, orother feature of pathology. For example, the degree of EGFR activationor activity or amount or number of EGFR positive cells, particularly ofantibody or binding member reactive or positive cells may be reduced.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined Tm with washes of higherstringency, if desired.

The present invention provides a novel specific binding member,particularly an antibody or fragment thereof, including immunogenicfragments, which recognizes an EGFR epitope which is found intumorigenic, hyperproliferative or abnormal cells wherein the epitope isenhanced or evident upon aberrant post-translational modification andnot detectable in normal or wild-type cells. In a particular butnonlimiting embodiment, the binding member, such as the antibody,recognizes an EGFR epitope which is enhanced or evident upon simplecarbohydrate modification or early glycosylation and is reduced or notevident in the presence of complex carbohydrate modification orglycosylation. The specific binding member, such as the antibody orfragment thereof, does not bind to or recognize normal or wild-typecells containing normal or wild-type EGFR epitope in the absence ofoverexpression and in the presence of normal EGFR post-translationalmodification.

The present invention further provides novel antibodies 806, 175, 124,1133, ch806, and hu806 and fragment thereof, including immunogenicfragments, which recognizes an EGFR epitope, particularly the EGFRpeptide (₂₈₇CGADSYEMEEDGVRKC₃₀₂ (SEQ ID NO:14)), which is exposed intumorigenic, hyperproliferative or abnormal cells wherein the epitope isenhanced, revealed, or evident and not detectable in normal or wild-typecells. In a particular but non-limiting embodiment, the antibodyrecognizes an EGFR epitope which is enhanced or evident upon simplecarbohydrate modification or early glycosylation and is reduced or notevident in the presence of complex carbohydrate modification orglycosylation. The antibody or fragment thereof does not bind to orrecognize normal or wild-type cells containing normal or wild-type EGFRepitope in the absence of overexpression, amplification, or atumorigenic event.

In a particular aspect of the invention and as stated above, the presentinventors have discovered the novel monoclonal antibodies 806, 175, 124,1133, 585, ch806, and hu806 which specifically recognize overexpressedwild-type EGFR and the de2-7 EGFR, yet bind to an epitope distinct fromthe unique junctional peptide of the de2-7 EGFR mutation. Additionally,while mAb806, mAb175, mAb124, mAb1133, and hu806 do not recognize thenormal, wild-type EGFR expressed on the cell surface of glioma cells,they do bind to the extracellular domain of the EGFR immobilized on thesurface of ELISA plates, indicating a conformational epitope with apolypeptide aspect.

Importantly, mAb806, mAb175, mAb124, mAb1133, mAb585, ch806, and hu806do not bind significantly to normal tissues such as liver and skin,which express levels of endogenous wtEGFR that are higher than in mostother normal tissues, but wherein EGFR is not overexpressed oramplified. Thus, mAb806, mAb175, mAb124, mAb1133, and hu806 demonstratenovel and useful specificity, recognizing de2-7 EGFR and overexpressedEGFR, while not recognizing normal, wild-type EGFR or the uniquejunctional peptide which is characteristic of de2-7 EGFR. In a preferredaspect mAb806, mAb175, mAb124, mAb1133, and hu806 of the presentinvention comprises the VH and VL chain CDR domain amino acid sequencesdepicted in FIGS. 14B and 15B; 74B and 75B; 51B and 51D; 52B and 52D;and 55A and 55B, respectively (SEQ ID NOS:2 and 4; 129 and 134; 22 and27; 32 and 37; and 42 and 47, respectively; SEQ ID NO:42 including thehu806 VH chain signal peptide and VH chain sequences of SEQ ID NOS:163and 164, respectively, and SEQ ID NO:47 including the hu806 VL chainsignal peptide and VL chain sequences of SEQ ID NOS: 165 and 166,respectively).

In another aspect, the invention provides an antibody capable ofcompeting with the 175 antibody, under conditions in which at least 10%of an antibody having the VH and VL chain sequences of the 175 antibody(SEQ ID NOS:129 and 134, respectively) is blocked from binding tode2-7EGFR by competition with such an antibody in an ELISA assay. As setforth above, anti-idiotype antibodies are contemplated herein.

The present invention relates to specific binding members, particularlyantibodies or fragments thereof, which recognizes an EGFR epitope whichis present in cells expressing overexpressed EGFR or expressing thede2-7 EGFR and not detectable in cells expressing normal or wild-typeEGFR, particularly in the presence of normal posttranslationalmodification.

It is further noted and herein demonstrated that an additionalnon-limiting observation or characteristic of the antibodies of thepresent invention is their recognition of their epitope in the presenceof high mannose groups, which is a characteristic of early glycosylationor simple carbohydrate modification. Thus, altered or aberrantglycosylation facilitates the presence and/or recognition of theantibody epitope or comprises a portion of the antibody epitope.

Glycosylation includes and encompasses the post-translationalmodification of proteins, termed glycoproteins, by addition ofoligosaccarides. Oligosaccharides are added at glycosylation sites inglycoproteins, particularly including N-linked oligosaccharides and0-linked oligosaccharides. N-linked oligosaccharides are added to an Asnresidue, particularly wherein the Asn residue is in the sequenceN-X-S/T, where X cannot be Pro or Asp, and are the most common onesfound in glycoproteins. In the biosynthesis of N-linked glycoproteins, ahigh mannose type oligosaccharide (generally comprised of dolichol,N-Acetylglucosamine, mannose and glucose is first formed in theendoplasmic reticulum (ER). The high mannose type glycoproteins are thentransported from the ER to the Golgi, where further processing andmodification of the oligosaccharides normally occurs. 0-linkedoligosaccharides are added to the hydroxyl group of Ser or Thr residues.In 0-linked oligosaccharides, N Acetylglucosamine is first transferredto the Ser or Thr residue by N Acetylglucosaminyltransferase in the ER.The protein then moves to the Golgi where further modification and chainelongation occurs.

In a particular aspect of the invention and as stated above, the presentinventors have discovered novel monoclonal antibodies, exemplifiedherein by the antibodies designated mAb806 (and its chimeric ch806),mAb175, mAb124, mAb1133, mAb585, and hu806 which specifically recognizeoverexpressed wild-type EGFR and the de2-7 EGFR, yet bind to an epitopedistinct from the unique junctional peptide of the de2-7 EGFR mutation.The antibodies of the present invention specifically recognizeoverexpressed EGFR, including amplified EGFR and mutant EGFR(exemplified herein by the de2-7 mutation), particularly upon aberrantpost-translational modification. Additionally, while these antibodies donot recognize the normal, wild-type EGFR expressed on the cell surfaceof glioma cells, they do bind to the extracellular domain of the EGFRimmobilized on the surface of ELISA plates, indicating a conformationalepitope with a polypeptide aspect. Importantly, these antibodies do notbind significantly to normal tissues such as liver and skin, whichexpress levels of endogenous wtEGFR that are higher than in most othernormal tissues, but wherein EGFR is not overexpressed or amplified.Thus, these antibodies demonstrate novel and useful specificity,recognizing de2-7 EGFR and amplified EGFR, while not recognizing normal,wild-type EGFR or the unique junctional peptide which is characteristicof de2-7 EGFR.

In a preferred aspect, the antibodies are ones which have thecharacteristics of the antibodies which the inventors have identifiedand characterized, in particular recognizing overexpressed EGFR andde2-7EGFR. In particularly preferred aspects, the antibodies are mAb806,mAb175, mAb124, mAb1133, mAb585, and hu806 or active fragments thereof.In a further preferred aspect the antibody of the present inventioncomprises the VH and VL chain amino acid sequences depicted FIGS. 16 and17; 74B and 75B; 51B and 51D; 52B and 52D; and 55A and 55B,respectively.

Preferably the epitope of the specific binding member or antibody islocated within the region comprising residues 273-501 of the maturenormal or wild-type EGFR sequence, and preferably the epitope comprisesresidues 287-302 of the mature normal or wild-type EGFR sequence (SEQ IDNO:14). Therefore, also provided are specific binding proteins, such asantibodies, which bind to the de2-7 EGFR at an epitope located withinthe region comprising residues 273-501 of the EGFR sequence, andcomprising residues 287-302 of the EGFR sequence (SEQ ID NO:14). Theepitope may be determined by any conventional epitope mapping techniquesknown to the person skilled in the art. Alternatively, the DNA sequencesencoding residues 273-501 and 287-302 (SEQ ID NO:14) could be digested,and the resultant fragments expressed in a suitable host. Antibodybinding could be determined as mentioned above.

In particular, the member will bind to an epitope comprising residues273-501, and more specifically comprising residues 287-302 (SEQ IDNO:14), of the mature normal or wild-type EGFR. However other antibodieswhich show the same or a substantially similar pattern of reactivityalso form an aspect of the invention. This may be determined bycomparing such members with an antibody comprising the VH and VL chaindomains shown in SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37;and 42 and 47, respectively. The comparison will typically be made usinga Western blot in which binding members are bound to duplicate blotsprepared from a nuclear preparation of cells so that the pattern ofbinding can be directly compared.

In another aspect, the invention provides an antibody capable ofcompeting with mAb806 under conditions in which at least 10% of anantibody having the VH and VL chain sequences of one of such antibodiesis blocked from binding to de2-7EGFR by competition with such anantibody in an ELISA assay. As set forth above, anti-idiotype antibodiesare contemplated and are illustrated herein.

In another aspect, the invention provides an antibody capable ofcompeting with mAb175, mAb124, mAb1133, and/or mAb585 under conditionsin which at least 10% of an antibody having the VH and VL chainsequences of one of such antibodies is blocked from binding to de2-7EGFRby competition with such an antibody in an ELISA assay. As set forthabove, anti-idiotype antibodies are contemplated and are illustratedherein.

In another aspect, the invention provides an antibody capable ofcompeting with mAb806, mAb175, mAb124, mAb1133, mAb585, and/or hu806,under conditions in which at least 10% of an antibody having the VH andVL chain sequences of one of such antibodies is blocked from binding tode2-7EGFR by competition with such an antibody in an ELISA assay. As setforth above, anti-idiotype antibodies are contemplated and areillustrated herein.

An isolated polypeptide consisting essentially of the epitope comprisingresidues 273-501 and more specifically comprising residues 287-302 (SEQID NO:14) of the mature wild-type EGFR forms another aspect of thepresent invention. The peptide of the invention is particularly usefulin diagnostic assays or kits and therapeutically or prophylactically,including as an anti-tumor or anti-cancer vaccine. Thus compositions ofthe peptide of the present invention include pharmaceutical compositionand immunogenic compositions.

Diagnostic and Therapeutic Uses

The unique specificity of the specific binding members, particularlyantibodies or fragments thereof, of the present invention, whereby thebinding member (s) recognize an EGFR epitope which is found intumorigenic, hyperproliferative or abnormal cells and not detectable innormal or wild-type cells and wherein the epitope is enhanced or evidentupon aberrant post-translational modification and wherein the member (s)bind to the de2-7 EGFR and overexpressed EGFR but not the wtEGFR,provides diagnostic and therapeutic uses to identify, characterize,target and treat, reduce or eliminate a number of tumorigenic cell typesand tumor types, for example head and neck, breast, lung, bladder orprostate tumors and glioma, without the problems associated with normaltissue uptake that may be seen with previously known EGFR antibodies.Thus, cells overexpressing EGFR (e.g. by amplification or expression ofa mutant or variant EGFR), particularly those demonstrating aberrantpost-translational modification may be recognized, isolated,characterized, targeted and treated or eliminated utilizing the bindingmember (s), particularly antibody (ies) or fragments thereof of thepresent invention.

In a further aspect of the invention, there is provided a method oftreatment of a tumor, a cancerous condition, a precancerous condition,and any condition related to or resulting from hyperproliferative cellgrowth comprising administration of mAb806, mAb175, mAb124, mAb1133,mAb585, and/or hu806.

The antibodies of the present invention can thus specifically categorizethe nature of EGFR tumors or tumorigenic cells, by staining or otherwiserecognizing those tumors or cells wherein EGFR overexpression,particularly amplification and/or EGFR mutation, particularly de2-7EGFR,is present. Further, the antibodies of the present invention, asexemplified by mAb806 (and chimeric antibody ch806), mAb175, mAb124,mAb1133, mAb585, and hu806, demonstrate significant in vivo anti-tumoractivity against tumors containing overexpressed EGFR and against de2-7EGFR positive xenografts.

As outlined above, the inventors have found that the specific bindingmember of the invention recognizes tumor-associated forms of the EGFR(de2-7 EGFR and overexpressed EGFR) but not the normal, wild-typereceptor when expressed in normal cells. It is believed that antibodyrecognition is dependent upon an aberrant posttranslational modification(e.g., a unique glycosylation, acetylation or phosphorylation variant)of the EGFR expressed in cells exhibiting overexpression of the EGFRgene.

As described below, antibodies of the present invention have been usedin therapeutic studies and shown to inhibit growth of overexpressing(e.g. amplified) EGFR xenografts and human de2-7 EGFR expressingxenografts of human tumors and to induce significant necrosis withinsuch tumors.

Moreover, the antibodies of the present invention inhibit the growth ofintracranial tumors in a preventative model. This model involvesinjecting glioma cells expressing de2-7 EGFR into nude mice and theninjecting the antibody intracranially either on the same day or within 1to 3 days, optionally with repeated doses. The doses of antibody aresuitably about 10 μg. Mice injected with antibody are compared tocontrols, and it has been found that survival of the treated mice issignificantly increased.

Therefore, in a further aspect of the invention, there is provided amethod of treatment of a tumor, a cancerous condition, a precancerouscondition, and any condition related to or resulting fromhyperproliferative cell growth comprising administration of a specificbinding member of the invention.

Antibodies of the present invention are designed to be used in methodsof diagnosis and treatment of tumors in human or animal subjects,particularly epithelial tumors. These tumors may be primary or secondarysolid tumors of any type including, but not limited to, glioma, breast,lung, prostate, head or neck tumors.

Binding Member and Antibody Generation

The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammering et al., “Monoclonal Antibodies And T cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; and 4,493,890.

Panels of monoclonal antibodies produced against EGFR can be screenedfor various properties; i.e., isotype, epitope, affinity, etc. Ofparticular interest are monoclonal antibodies that mimic the activity ofEGFR or its subunits. Such monoclonals can be readily identified inspecific binding member activity assays. High affinity antibodies arealso useful when immunoaffinity purification of native or recombinantspecific binding member is possible.

Methods for producing polyclonal anti-EGFR antibodies are well-known inthe art. See U.S. Pat. No. 4,493,795 to Nestor et al. A monoclonalantibody, typically containing Fab and/or F (ab′) 2 portions of usefulantibody molecules, can be prepared using the hybridoma technologydescribed in Antibodies-A Laboratory Manual, Harlow and Lane, eds., ColdSpring Harbor Laboratory, New York (1988), which is incorporated hereinby reference. Briefly, to form the hybridoma from which the monoclonalantibody composition is produced, a myeloma or other self-perpetuatingcell line is fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with an appropriate EGFR.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody useful in practicingthis invention are identified by their ability to immunoreact with thepresent antibody or binding member and their ability to inhibitspecified tumorigenic or hyperproliferative activity in target cells.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-EGFR antibodies are alsowell-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA,80:4949-4953 (1983). Typically, the EGFR or a peptide analog is usedeither alone or conjugated to an immunogenic carrier, as the immunogenin the before described procedure for producing anti-EGFR monoclonalantibodies. The hybridomas are screened for the ability to produce anantibody that immunoreacts with the EGFR present in tumorigenic,abnormal or hyperproliferative cells. Other anti-EGFR antibodies includebut are not limited to the HuMAX-EGFr antibody from Genmab/Medarex, the108 antibody (ATCC HB9764) and U.S. Pat. No. 6,217,866, and antibody14E1 from Schering AG (U.S. Pat. No. 5,942,602).

Recombinant Binding Members, Chimerics, Bispecifics and Fragments

In general, the CDR1 regions, comprising amino acid sequencessubstantially as set out as the CDR1 regions of SEQ ID NOS:2 and 4; 129and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will becarried in a structure which allows for binding of the CDR1 regions toan tumor antigen. In the case of the CDR1 region of SEQ ID NO:4, forexample, this is preferably carried by the VL chain region of SEQ IDNO:4 (and similarly for the other recited sequences).

In general, the CDR2 regions, comprising amino acid sequencessubstantially as set out as the CDR2 regions of SEQ ID NOS:2 and 4; 129and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will becarried in a structure which allows for binding of the CDR2 regions toan tumor antigen. In the case of the CDR2 region of SEQ ID NO:4, forexample, this is preferably carried by the VL chain region of SEQ IDNO:4 (and similarly for the other recited sequences).

In general, the CDR3 regions, comprising amino acid sequencessubstantially as set out as the CDR3 regions of SEQ ID NOS:2 and 4; 129and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will becarried in a structure which allows for binding of the CDR3 regions toan tumor antigen. In the case of the CDR3 region of SEQ ID NO:4, forexample, this is preferably carried by the VL chain region of SEQ IDNO:4 (and similarly for the other recited sequences).

By “substantially as set out” it is meant that that CDR regions, forexample CDR3 regions, of the invention will be either identical orhighly homologous to the specified regions of SEQ ID NOS:2 and 4; 129and 134; 22 and 27; 32 and 37; and 42 and 47, respectively. By “highlyhomologous” it is contemplated that only a few substitutions, preferablyfrom 1 to 8, preferably from 1 to 5, preferably from 1 to 4, or from 1to 3 or 1 or 2 substitutions may be made in one or more of the CDRs. Itis also contemplated that such terms include truncations to the CDRs, solong as the resulting antibody exhibits the unique properties of theclass of antibodies discussed herein, as exhibited by mAb806, mAb175,mAb124, mAb1133 and hu806.

The structure for carrying the CDRs of the invention, in particularCDR3, will generally be of an antibody heavy or light chain sequence orsubstantial portion thereof in which the CDR regions are located atlocations corresponding to the CDR region of naturally occurring VH andVL chain antibody variable domains encoded by rearranged immunoglobulingenes. The structures and locations of immunoglobulin variable domainsmay be determined by reference to Kabat, E. A. et al, Sequences ofProteins of Immunological Interest. 4th Edition. US Department of Healthand Human Services. 1987, and updates thereof, now available on theInternet (http://immuno.bme.nwu.edu)). Moreover, as is known to those ofskill in the art, CDR determinations can be made in various ways. Forexample, Kabat, Chothia and combined domain determination analyses maybe used. In this regard, see for examplehttp://www.bioinf.org.uk/abs/#cdrid.

Preferably, the amino acid sequences substantially as set out as the VHchain CDR residues in the inventive antibodies are in a human heavychain variable domain or a substantial portion thereof, and the aminoacid sequences substantially as set out as the VL chain CDR residues inthe inventive antibodies are in a human light chain variable domain or asubstantial portion thereof.

The variable domains may be derived from any germline or rearrangedhuman variable domain, or may be a synthetic variable domain based onconsensus sequences of known human variable domains. The CDR3-derivedsequences of the invention, for example, as defined in the precedingparagraph, may be introduced into a repertoire of variable domainslacking CDR3 regions, using recombinant DNA technology.

For example, Marks et al (Bio/Technology, 1992,10:779-783) describemethods of producing repertoires of antibody variable domains in whichconsensus primers directed at or adjacent to the 5′ end of the variabledomain area are used in conjunction with consensus primers to the thirdframework region of human VH genes to provide a repertoire of VHvariable domains lacking a CDR3. Marks et al further describe how thisrepertoire may be combined with a CDR3 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide specific binding members of the invention.The repertoire may then be displayed in a suitable host system such asthe phage display system of W092/01047 so that suitable specific bindingmembers may be selected. A repertoire may consist of from anything from10⁴ individual members upwards, for example from 10⁶ to 10⁸ or 10¹⁰members.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature, 1994, 370:389-391), who describes the technique inrelation to a p-lactamase gene but observes that the approach may beused for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying theCDR3 derived sequences of the invention using random mutagenesis of, forexample, the mAb806 VH or VL genes to generate mutations within theentire variable domain. Such a technique is described by Gram et al(1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-pronePCR.

Another method which may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al, (1994,Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al. (1996, J.Mol. Biol. 263:551-567).

All the above described techniques are known as such in the art and inthemselves do not form part of the present invention. The skilled personwill be able to use such techniques to provide specific binding membersof the invention using routine methodology in the art.

A substantial portion of an immunoglobulin variable domain will compriseat least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of specific bindingmembers of the present invention made by recombinant DNA techniques mayresult in the introduction of N- or C-terminal residues encoded bylinkers introduced to facilitate cloning or other manipulation steps.Other manipulation steps include the introduction of linkers to joinvariable domains of the invention to further protein sequences includingimmunoglobulin heavy chains, other variable domains (for example in theproduction of diabodies) or protein labels as discussed in more detailbelow.

Although in a preferred aspect of the invention specific binding memberscomprising a pair of binding domains based on sequences substantiallyset out in SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42and 47, respectively, are preferred, single binding domains based onthese sequences form further aspects of the invention. In the case ofthe binding domains based on the sequence substantially set out in VHchains, such binding domains may be used as targeting agents for tumorantigens since it is known that immunoglobulin VH domains are capable ofbinding target antigens in a specific manner.

In the case of either of the single chain specific binding domains,these domains may be used to screen for complementary domains capable offorming a two-domain specific binding member which has in vivoproperties as good as or equal to the mAb806, ch806, mAb175, mAb124,mAb1133, mAb585, and hu806 antibodies disclosed herein.

This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed in U.S.Pat. No. 5,969,108 in which an individual colony containing either an Hor L chain clone is used to infect a complete library of clones encodingthe other chain (L or H) and the resulting two-chain specific bindingmember is selected in accordance with phage display techniques such asthose described in that reference. This technique is also disclosed inMarks et al, ibid.

Specific binding members of the present invention may further compriseantibody constant regions or parts thereof. For example, specificbinding members based on VL chain sequences may be attached at theirC-terminal end to antibody light chain constant domains including humanCk of Cλ chains, preferably Cλ chains. Similarly, specific bindingmembers based on VH chain sequences may be attached at their C-terminalend to all or part of an immunoglobulin heavy chain derived from anyantibody isotype, e.g. IgG, IgA, IgE, IgD and IgM and any of the isotypesub-classes, particularly IgGl, IgG2b, and IgG4. IgG1 is preferred.

The advent of monoclonal antibody (mAb) technology 25 years ago hasprovide an enormous repertoire of useful research reagents and createdthe opportunity to use antibodies as approved pharmaceutical reagents incancer therapy, autoimmune disorders, transplant rejection, antiviralprophylaxis and as anti-thrombotics (Glennie and Johnson, 2000). Theapplication of molecular engineering to convert murine mAbs intochimeric mAbs (mouse V-region, human C-region) and humanized reagentswhere only the mAb complementarity-determining regions (CDR) are ofmurine origin has been critical to the clinical success of mAb therapy.The engineered mAbs have markedly reduced or absent immunogenicity,increased serum half-life and the human Fc portion of the mAb increasesthe potential to recruit the immune effectors of complement andcytotoxic cells (Clark 2000). Investigations into the biodistribution,pharmacokinetics and any induction of an immune response to clinicallyadministered mAbs requires the development of analyses to discriminatebetween the pharmaceutical and endogenous proteins.

The antibodies, or any fragments thereof, may also be conjugated orrecombinantly fused to any cellular toxin, bacterial or other, e.g.pseudomonas exotoxin, ricin, or diphtheria toxin. The part of the toxinused can be the whole toxin, or any particular domain of the toxin. Suchantibody-toxin molecules have successfully been used for targeting andtherapy of different kinds of cancers, see e.g. Pastan, Biochim BiophysActa. 1997 Oct. 24; 1333 (2):C1-6; Kreitman et al., N. Engl. J. Med.2001 Jul. 26; 345 (4):241-7; Schnell et al., Leukemia. 2000 January; 14(1):129-35; Ghetie et al., Mol. Biotechnol. 2001 Jul; 18 (3):251-68.

Bi-and tri-specific multimers can be formed by association of differentscFv molecules and have been designed as cross-linking reagents forT-cell recruitment into tumors (immunotherapy), viral retargeting (genetherapy) and as red blood cell agglutination reagents(immunodiagnostics), see e.g. Todorovska et al., J. Immunol. Methods.2001 Feb. 1; 248 (1-2):47-66; Tomlinson et al., Methods Enzymol. 2000;326:461-79; McCall et al., J. Immunol. 2001 May 15; 166 (10):6112-7.

Fully human antibodies can be prepared by immunizing transgenic micecarrying large portions of the human immunoglobulin heavy and lightchains. These mice, examples of such mice are the Xenomouse™ (Abgenix,Inc.) (U.S. Pat. Nos. 6,075,181 and 6,150,584), the HuMAb-Mouse™(Medarex, Inc./GenPharm) (U.S. Pat. Nos. 5,545,806 and 5,569,825), theTransChromo Mouse (Kirin) and the KM Mouse (Medarex/Kirin), are wellknown within the art.

Antibodies can then be prepared by, e.g. standard hybridoma technique orby phage display. These antibodies will then contain only fully humanamino acid sequences.

Fully human antibodies can also be generated using phage display fromhuman libraries. Phage display may be performed using methods well knownto the skilled artisan, as in Hoogenboom et al. and Marks et al.(Hoogenboom H R and Winter G. (1992) J. Mol. Biol. 227 (2):381-8; MarksJ D et al. (1991) J. Mol. Biol. 222 (3):581-97; and also U.S. Pat. Nos.5,885,793 and 5,969,108).

Therapeutic Antibodies and Uses

The in vivo properties, particularly with regard to tumor:blood ratioand rate of clearance, of specific binding members of the invention willbe at least comparable to mAb806. Following administration to a human oranimal subject such a specific binding member will show a peak tumor toblood ratio of >1:1. Preferably at such a ratio the specific bindingmember will also have a tumor to organ ratio of greater than 1:1,preferably greater than 2:1, more preferably greater than 5:1.Preferably at such a ratio the specific binding member will also have anorgan to blood ratio of <1:1 in organs away from the site of the tumor.These ratios exclude organs of catabolism and secretion of theadministered specific binding member. Thus in the case of scFvs and Fabs(as shown in the accompanying examples), the binding members aresecreted via the kidneys and there is greater presence here than otherorgans. In the case of whole IgGs, clearance will be at least in part,via the liver. The peak localization ratio of the intact antibody willnormally be achieved between 10 and 200 hours following administrationof the specific binding member. More particularly, the ratio may bemeasured in a tumor xenograft of about 0.2-1.0 g formed subcutaneouslyin one flank of an athymic nude mouse.

Antibodies of the invention may be labelled with a detectable orfunctional label. Detectable labels include, but are not limited to,radiolabels such as the isotopes 3H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co,⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹¹¹In, ²¹¹At, ¹⁹⁸Au, ⁶⁷CU,²²⁵Ac, ²¹³Bi, ⁹⁹Tc and ¹⁸⁶Rc, which may be attached to antibodies of theinvention using conventional chemistry known in the art of antibodyimaging. Labels also include fluorescent labels and labels usedconventionally in the art for MRI-CT imagine. They also include enzymelabels such as horseradish peroxidase. Labels further include chemicalmoieties such as biotin which may be detected via binding to a specificcognate detectable moiety, e.g. labeled avidin.

Functional labels include substances which are designed to be targetedto the site of a tumor to cause destruction of tumor tissue. Suchfunctional labels include cytotoxic drugs such as 5-fluorouracil orricin and enzymes such as bacterial carboxypeptidase or nitroreductase,which are capable of converting prodrugs into active drugs at the siteof a tumor.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of the specificbinding members, antibodies and/or their subunits may possess certaindiagnostic applications and may for example, be utilized for the purposeof detecting and/or measuring conditions such as cancer, precancerouslesions, conditions related to or resulting from hyperproliferative cellgrowth or the like. For example, the specific binding members,antibodies or their subunits may be used to produce both polyclonal andmonoclonal antibodies to themselves in a variety of cellular media, byknown techniques such as the hybridoma technique utilizing, for example,fused mouse spleen lymphocytes and myeloma cells. Likewise, smallmolecules that mimic or antagonize the activity (ies) of the specificbinding members of the invention may be discovered or synthesized, andmay be used in diagnostic and/or therapeutic protocols.

The radiolabeled specific binding members, particularly antibodies andfragments thereof, are useful in in vitro diagnostics techniques and inin vivo radioimaging techniques and in radioimmunotherapy. In theinstance of in vivo imaging, the specific binding members of the presentinvention may be conjugated to an imaging agent rather than aradioisotope (s), including but not limited to a magnetic resonanceimage enhancing agent, wherein for instance an antibody molecule isloaded with a large number of paramagnetic ions through chelatinggroups. Examples of chelating groups include EDTA, porphyrins,polyamines crown ethers and polyoximes. Examples of paramagnetic ionsinclude gadolinium, iron, manganese, rhenium, europium, lanthanium,holmium and erbium. In a further aspect of the invention, radiolabeledspecific binding members, particularly antibodies and fragments thereof,particularly radioimmunoconjugates, are useful in radioimmunotherapy,particularly as radiolabeled antibodies for cancer therapy. In a stillfurther aspect, the radiolabelled specific binding members, particularlyantibodies and fragments thereof, are useful in radioimmuno-guidedsurgery techniques, wherein they can identify and indicate the presenceand/or location of cancer cells, precancerous cells, tumor cells, andhyperproliferative cells, prior to, during or following surgery toremove such cells.

The present invention also includes immunoconjugates, wherein specificbinding members of the present invention, particularly antibodies andfragments thereof, are conjugated or attached to one or more agents formodifying a biological response (such as, for example and withoutlimitation, inhibiting or preventing the expression activity of cells,causing the destruction of cells, or otherwise effecting the function ofcells). Such agents include, for example and without limitation,chemical ablation agents, toxins, immunomodulators, cytokines, cytotoxicagents, chemotherapeutic agents and/or drugs, and include, but are notlimited to, the following:

-   -   4-desacetylvinblastine-3-carbohydiazide;    -   5-fluoro-2′-deoxyuridine;    -   5-fluorouracil;    -   5-fluorouracil decarbonizes;    -   6-mercaptopurine;    -   6-thioguanine;    -   abrin;    -   abrin A chain;    -   actinomycin D;    -   actinomycin D, 1-dehydrotestosterone;    -   adriamycin;    -   alkylating agents;    -   alkylphosphocholines;    -   aminopterin;    -   angiogenin;    -   angiostatin;    -   anthracyclines;    -   anthramycin;    -   anti-angiogenics;    -   anti-folates;    -   anti-metabolites;    -   anti-mitotics;    -   antibiotics;    -   ara-C;    -   auristatin derivatives (see, for example and without limitation,        U.S. Patent Publication Nos. 2008/0300192, 2009/0018086,        2009/0018086, and 2009/0111756, each of which is hereby        incorporated by reference in its entirety);    -   auristatin E (see, for example and without limitation, U.S. Pat.        No. 5,635,483, hereby incorporated by reference in its        entirety);    -   auristatin E valeryl benzylhydrazone;    -   auristatin F phenylene diamine;    -   auristatins;    -   auromycins;    -   bis-iodo-phenol mustard;    -   bismuth;    -   bleomycin;    -   busulfan;    -   calicheamicin;    -   carboplatin;    -   carminomycin;    -   carmustine;    -   cc-1065 compounds (see, for example and without limitation, U.S.        Pat. Nos. 5,475,092, 5,585,499, 5,846,545, 6,534,660, 6,586,618,        6,756,397, 7,049,316, 7,329,760, 7,388,026, 7,655,660, and        7,655,661, U.S. Patent Publication. Nos. 2007/0135346,        2008/0260685, and 2009/0281158, and 2009/0318668, and PCT        Publication No. WO2009/017394, each of which is hereby        incorporated by reference in its entirety);    -   chlorambucil;    -   cis-dichlorodiamine platinum (cisplatin);    -   colchicin (colchicine);    -   combrestatin;    -   crotin;    -   curicin;    -   cyclothosphamide;    -   cytarabine;    -   cytochalasin B;    -   cytosine arabinoside;    -   cytoxin;    -   dacarbazine;    -   dactinomycin (actinomycin);    -   daunorubicin (daunomycin);    -   dibromomannitol;    -   dihydroxy anthracin dione;    -   diphtheria toxin;    -   dolastatin-10;    -   doxetaxel;    -   doxorubicin;    -   doxorubicin hydrazides;    -   duocarmycins (see, for example and without limitation, U.S. Pat.        No. 7,214,685, hereby incorporated by reference in its        entirety);    -   emetine;    -   endostatin;    -   enediyenes;    -   enomycin;    -   epirubicin;    -   esperamicin compounds (see, for example and without limitation,        U.S. Pat. No. 4,675,187, hereby incorporated by reference in its        entirety);    -   ethidium bromide;    -   etoposide;    -   gelonin;    -   glucocorticoids;    -   gramicidin D;    -   granulocyte colony stimulating factor;    -   granulocyte macrophage colony stimulating factor;    -   idarubicin;    -   intercalating agents;    -   interleukin-1;    -   interleukin-2;    -   interleukin-6;    -   lidocaine;    -   lomustine;    -   lymphokine;    -   maytansinols (see, for example and without limitation, U.S. Pat.        Nos. 4,137,230, 4,151,042, 4,162,940, 4,190,580, 4,225,494,        4,228,239, 4,248,870, 4,256,746, 4,260,608, 4,263,294,        4,264,596, 4,265,814, 4,294,757, 4,307,016, 4,308,268,        4,308,269, 4,309,428, 4,317,821, 4,320,200, 4,322,348,        4,331,598, 4,360,462, 4,361,650, 4,362,663, 4,364,866,        4,371,533, 4,424,219, 4,450,234, 5,141,736, and 5,217,713, each        of which is hereby incorporated by reference in its entirety);    -   mechlorethamine;    -   melphalan (and other related nitrogen mustards);    -   methotrexate;    -   minor groove-binders;    -   mithramycin;    -   mitogellin;    -   mitomycin C;    -   mitomycins;    -   mitoxantrone;    -   MMAF-dimethylaminoethylamine;    -   MMAF-N-t-butyl;    -   MMAF-tetraethylene glycol;    -   modeccin A chain;    -   mono-methyl auristatin E (MMAE) (see, for example and without        limitation, U.S. Pat. Nos. 6,884,869, 7,098,308, 7,256,257, and        7,423,116, and U.S. Patent Publication Nos. 2003/0083263,        2004/0157782, 2005/0009751, 2005/0113308, and 2006/0229253, each        of which is hereby incorporated by reference in its entirety);    -   mono-methyl auristatin F (MMAF) (see, for example and without        limitation, U.S. Pat. No. 7,498,298, and U.S. Patent Publication        Nos. 2008/0226657, 2008/0248051, 2008/0248053, and 2009/0047296,        each of which is hereby incorporated by reference in its        entirety);    -   morpholinodoxorubicin;    -   N2′-deacetyl-N2′-(c-mercapto-1 oxopropyl)-maytansine (DM1) (see,        for example and without limitation, U.S Pat. No. 5,208,020,        hereby incorporated by reference in its entirety);    -   N2′-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine        (DM4) (see, for example and without limitation, U.S. Pat. No.        7,276,497, hereby incorporated by reference in its entirety);    -   neocarzinostatin;    -   nerve growth factor (and other growth factors);    -   onapristone;    -   paclitaxel;    -   PE40;    -   phenomycin;    -   platelet derived growth factor;    -   prednisone;    -   procaine;    -   propranolol;    -   Pseudomonas exotoxin A;    -   puromycin;    -   radioactive isotopes (such as, for example and without        limitation, At²¹¹, Bi²¹², Bi²¹³, Cf²⁵², I¹²⁵, I¹³¹, In¹¹¹,        Ir¹⁹², Lu¹⁷⁷, P³², Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Y⁹⁰, and W¹⁸⁸);    -   retstrictocin;    -   ricin A;    -   ricins;    -   Sapaonaria officinalis inhibitor;    -   saporin;    -   streptozotocin;    -   suramin;    -   tamoxifen;    -   taxanes;    -   taxoids;    -   taxol;    -   tenoposide;    -   tetracaine;    -   thioepa chlorambucil;    -   thiotepa;    -   thrombotic agents;    -   tissue plasminogen activator;    -   topoisomerase I inhibitors;    -   topoisomerase II inhibitors;    -   toxotere;    -   tumor necrosis factor;    -   vinblastine;    -   vinca alkaloids;    -   vincas;    -   vincristine;    -   vindesine;    -   vinorelbine;    -   yttrium;    -   α-interferon;    -   α-sarcin; and    -   β-interferon,        as well as analogs, homologs, fragments, variants, and        derivatives thereof (see also Garnett (2001) Advanced drug        Delivery Reviews 53:171-216, hereby incorporated by reference in        its entirety).

As will be understood by those of skill in the art, the agents set forthabove, as well as other suitable agents, may be conjugated or attachedto specific binding members of the present invention, particularlyantibodies and fragments thereof, in any suitable manner to produceimmunoconjugates of the present invention. For example and withoutlimitation, in various embodiments of the present invention the bindingmember(s) and agent(s) may be covalently attached and/or may beconjugated using linker, spacer and/or stretcher compounds, which invarious embodiments of the present invention are cleavable, arenon-cleavable, and result in the therapeutic agent(s) being internalizedby the target cell.

For example, such linker, spacer and/or stretcher compounds include, butare not limited to, the following: amino benzoic acid spacers (see, forexample and without limitation, U.S. Pat. Nos. 7,091,186 and 7,553,816,each of which is hereby incorporated by reference in its entirety);maleimidocaproyl; p-aminobenzylcarbamoyl (PAB); lysosomalenzyme-cleavable linkers (see, for example and without limitation, U.S.Pat. No. 6,214,345, hereby incorporated by reference in its entirety);maleimidocaproyl-polyethylene glycol (MC(PEG)6-OH); N-methyl-valinecitrulline; N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (see, for exampleand without limitation, Yoshitake et al. (1979) Eur. J. Biochem., 101,395-399, hereby incorporated by reference in its entirety);N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, for example andwithout limitation, U.S. Pat. No. 4,563,304, hereby incorporated byreference in its entirety); N-Succinimidyl 4-(2-pyridylthio)pentanoate(SPP); valine-citrulline; and other linker, spacer, and/or stretchercompounds (see, for example and without limitation, U.S. Pat. Nos.7,090,843, 7,223,837, and 7,659,241, and U.S. Patent Publication Nos.2004/0018194, 2004/0121940, 2006/0116422, 2007/0258987, 2008/0213289,2008/0241128, 2008/0311136, 2008/0317747, and 2009/0010945, each ofwhich is hereby incorporated by reference in its entirety).

Generally speaking, techniques for attaching and/or conjugating theagents set forth above, as well as other agents, to specific bindingmembers of the present invention, particularly antibodies and fragmentsthereof, are known in the art. See, for example and without limitation,Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs InCancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeldet al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,“Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.),Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, inMonoclonal Antibodies '84: Biological And Clinical Applications,Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, AndFuture Prospective Of The Therapeutic Use Of Radiolabeled Antibody InCancer Therapy”, in Monoclonal Antibodies For Cancer Detection AndTherapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), andThorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982), each ofwhich is hereby incorporated by reference in its entirety.

Furthermore, specific binding members of the present invention,particularly antibodies and fragments thereof, may be conjugated to asecond antibody to form an antibody heteroconjugate (see, for exampleand without limitation, U.S. Pat. No. 4,676,980, hereby incorporated byreference in its entirety), may be administered (either with or withoutan agent attached or conjugated thereto) alone or in combination withanother agent (for example and without limitation, an agent set forthabove), and/or may be conjugated to an anti-cancer pro-drug activatingenzyme capable of converting the pro-drug to its active form.

Radioimmunotherapy (RAIT) has entered the clinic and demonstratedefficacy using various antibody immunoconjugates. ¹³¹1 labeled humanizedanti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 has beenevaluated in colorectal cancer (Behr T M et al (2002) Cancer 94(4Suppl):1373-81) and the same antibody with 90Y label has been assessedin medullary thyroid carcinoma (Stein R et al (2002) Cancer 94(1):51-61). Radioimmunotherapy using monoclonal antibodies has also beenassessed and reported for non-Hodgkin's lymphoma and pancreatic cancer(Goldenberg D M (2001) Crit. Rev. Oncol. Hematol. 39 (1-2):195-201; GoldD V et al. (2001) Crit. Rev. Oncol. Hematol. 39 (1-2) 147-54).Radioimmunotherapy methods with particular antibodies are also describedin U.S. Pat. Nos. 6,306,393 and 6,331,175. Radioimmunoguided surgery(RIGS) has also entered the clinic and demonstrated efficacy andusefulness, including using anti-CEA antibodies and antibodies directedagainst tumor-associated antigens (Kim J C et al (2002) Jut. J. Cancer97(4):542-7; Schneebaum, S. et al. (2001) World J. Surg. 25(12):1495-8;Avital, S. et al. (2000) Cancer 89(8):1692-8; McIntosh D G et al (1997)Cancer Biother. Radiopharm. 12 (4):287-94).

Antibodies of the present invention may be administered to a patient inneed of treatment via any suitable route, usually by injection into thebloodstream or CSF, or directly into the site of the tumor. The precisedose will depend upon a number of factors, including whether theantibody is for diagnosis or for treatment, the size and location of thetumor, the precise nature of the antibody (whether whole antibody,fragment, diabody, etc.), and the nature of the detectable or functionallabel attached to the antibody. Where a radioisotope is used fortherapy, a suitable maximum single dose is about 45 mCi/m², to a maximumof about 250 mCi/m². Preferable dosage is in the range of 15 to 40 mCi,with a further preferred dosage range of 20 to 30 mCi, or 10 to 30 mCi,depending on the isotope utilized. Such therapy may require bone marrowor stem cell replacement. A typical antibody dose for either tumorimaging or radioisotope-conjugated tumor treatment will be in the rangeof from 0.5 to 1000 mg. Naked antibodies are preferably administered indoses of 20 to 1000 mg protein per dose, or 20 to 500 mg protein perdose, or 20 to 100 mg protein per dose. This is a dose for a singletreatment of an adult patient, which may be proportionally adjusted forchildren and infants, and also adjusted for other antibody formats inproportion to molecular weight. Treatments may be repeated at daily,twice-weekly, weekly or monthly intervals, at the discretion of thephysician.

These formulations may include a second binding protein, such as theEGPR binding proteins described supra. In an especially preferred form,this second binding protein is a monoclonal antibody such as 528 or 225,discussed infra.

Pharmaceutical and Therapeutic Compositions

Specific binding members of the present invention will usually beadministered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the specific bindingmember.

Thus pharmaceutical compositions according to the present invention, andfor use in accordance with the present invention, may comprise, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabilizer or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be included, as required.

A specific binding member, antibody or fragment thereof, or acomposition comprising a specific binding member, antibody or fragmentthereof, of the present invention, may be administered alone or incombination with other treatments, therapeutics or agents, dependingupon the condition to be treated. In addition, the present inventioncontemplates and includes compositions comprising the binding member,particularly an antibody or fragment thereof, herein described and otheragents or therapeutics such as anti-cancer agents or therapeutics,hormones, anti-EGFR agents or antibodies, or immune modulators. Moregenerally, these anti-cancer agents may be tyrosine kinase inhibitors orphosphorylation cascade inhibitors, post-translational modulators, cellgrowth or division inhibitors (e.g. anti-mitotics), or signaltransduction inhibitors. Other treatments or therapeutics may includethe administration of suitable doses of pain relief drugs such asnon-steroidal anti-inflammatory drugs (e.g., aspirin, paracetamol,ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics.

For example and without limitation, a specific binding member, antibodyor fragment thereof, or a composition comprising a specific bindingmember, antibody or fragment thereof, of the present invention, may beadministered in combination with a tyrosine kinase inhibitor (including,but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668),doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas,procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosinearabinoside, cyclophosphamide, epipodophyllotoxin, carmustine,lomustine, and/or other chemotherapeutic agents. Thus, these agents maybe anti-EGFR specific agents, or tyrosine kinase inhibitors such asAG1478, ZD1839, STI571, OSI-774, or SU-6668 or may be more generalanti-cancer and anti-neoplastic agents such as doxorubicin, cisplatin,temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea,5-fluoruracil, cytosine arabinoside, cyclophosphamide,epipodophyllotoxin, carmustine, or lomustine.

Furthermore, the specific binding member, antibody or fragment thereof,or composition comprising a specific binding member, antibody orfragment thereof, of the present invention, may be administered incombination with hormones such as dexamethasone, immune modulators, suchas interleukins, tumor necrosis factor (TNF) or other growth factors orcytokines which stimulate the immune response and reduction orelimination of cancer cells or tumors.

As used herein, administration of a specific binding member, antibody orfragment thereof, or a composition comprising a specific binding member,antibody or fragment thereof, of the present invention, in combinationwith other treatments, therapeutics or agents includes, for example andwithout limitation, sequential administration (i.e., before or after),simultaneous administration, and both sequential and simultaneousadministration.

It is to be understood that reference to a specific binding member,antibody or fragment thereof, or a composition comprising a specificbinding member, antibody or fragment thereof, of the present invention,includes, one or more specific binding members, antibodies or fragmentsthereof, and one or more compositions comprising one or more specificbinding members, antibodies or fragments thereof.

Both sequential and simultaneous administration refers, for example andwithout limitation, to administration of the specific binding member,antibody or fragment thereof, or composition comprising a specificbinding member, antibody or fragment thereof, of the present invention,prior to administration of another treatment, therapeutic or agent,followed by simultaneous administration of such specific binding member,antibody or fragment thereof, or composition, with a treatment,therapeutic or agent.

In all instances, administration of a specific binding member, antibodyor fragment thereof, or a composition comprising a specific bindingmember, antibody or fragment thereof, of the present invention, incombination with other treatments, therapeutics or agents refers, forexample and without limitation, to both a single treatment as well as tomultiple treatments, of the specific binding member, antibody orfragment thereof, or a composition comprising a specific binding member,antibody or fragment thereof, of the present invention, and the othertreatments, therapeutics or agents, independently or in combinationthereof.

Illustrative, non-limiting examples, of the manner in which a specificbinding member, antibody or fragment thereof, or a compositioncomprising a specific binding member, antibody or fragment thereof, ofthe present invention, may be administered in combination with othertreatments, therapeutics or agents, are provided in Examples 27-33herein.

An immune modulator such as TNF may be combined together with a memberof the invention in the form of a bispecific antibody recognizing theEGFR epitope recognized by the inventive antibodies, as well as bindingto TNF receptors. The composition may also be administered with, or mayinclude combinations along with other anti-EGFR antibodies, includingbut not limited to the anti-EGFR antibodies 528, 225, SC-03, DR8. 3,L8A4, Y10, ICR62 and ABX-EGF.

Previously the use of agents such as doxorubicin and cisplatin inconjunction with anti-EGFR antibodies have produced enhanced anti-tumoractivity (Fan et al, 1993; Baselga et al, 1993). The combination ofdoxorubicin and mAb 528 resulted in total eradication of establishedA431 xenografts, whereas treatment with either agent alone caused onlytemporary in vivo growth inhibition (Baselga et al, 1993). Likewise, thecombination of cisplatin and either mAb528 or 225 also led to theeradication of well established A431 xenografts, which was not observedwhen treatment with either agent was used (Fan et al, 1993).

Conventional Radiotherapy

In addition, the present invention contemplates and includes therapeuticcompositions for the use of the binding member in combination withconventional radiotherapy. It has been indicated that treatment withantibodies targeting EGF receptors can enhance the effects ofconventional radiotherapy (Milas et al., Clin. Cancer Res. 2000February: 6 (2):701, Huang et al., Clin. Cancer Res. 2000 June: 6(6):2166).

As demonstrated herein, combinations of the binding member of thepresent invention, particularly an antibody or fragment thereof,preferably the mAb806, ch806, mAb175, mAb124, mAb1133, mAb585, or hu806,or a fragment thereof, and anti-cancer therapeutics, particularlyanti-EGFR therapeutics, including other anti-EGFR antibodies,demonstrate effective therapy, and particularly synergy, againstxenografted tumors. In the Examples, it is demonstrated, for example,that the combination of AG1478 and mAb806 results in significantlyenhanced reduction of A431 xenograft tumor volume in comparison withtreatment with either agent alone. AG1478(4-(3-chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selectiveinhibitor of the EGF receptor kinase and is particularly described inU.S. Pat. No. 5,457,105, incorporated by reference herein in itsentirety (see also, Liu, W. et al (1999) J. Cell Sci. 112:2409; Eguchi,S. et al. (1998) J. Biol. Chem. 273:8890; Levitsky, A. and Gazit, A.(1995) Science 267:1782). The Specification Examples further demonstratetherapeutic synergy of antibodies of the present invention with otheranti-EGFR antibodies, particularly with the 528 anti-EGFR antibody.

The present invention further contemplates therapeutic compositionsuseful in practicing the therapeutic methods of this invention. Asubject therapeutic composition includes, in admixture, apharmaceutically acceptable excipient (carrier) and one or more of aspecific binding member, polypeptide analog thereof or fragment thereof,as described herein as an active ingredient. In a preferred embodiment,the composition comprises an antigen capable of modulating the specificbinding of the present binding member/antibody with a target cell.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions. However, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog-or active fragment-containingcompositions are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofEGFR binding capacity desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosages may rangefrom about 0.1 to 20, preferably about 0.5 to about 10, and morepreferably one to several, milligrams of active ingredient per kilogrambody weight of individual per day and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by repeated doses at one or more hour intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain concentrations of ten nanomolar to tenmicromolar in the blood are contemplated.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be included, as required.

Diagnostic Assays

The present invention also relates to a variety of diagnosticapplications, including methods for detecting the presence of stimulisuch as aberrantly expressed EGFR, by reference to their ability to berecognized by the present specific binding member. As mentioned earlier,the EGFR can be used to produce antibodies to itself by a variety ofknown techniques, and such antibodies could then be isolated andutilized as in tests for the presence of particular EGFR activity insuspect target cells.

Diagnostic applications of the specific binding members of the presentinvention, particularly antibodies and fragments thereof, include invitro and in vivo applications well known and standard to the skilledartisan and based on the present description. Diagnostic assays and kitsfor in vitro assessment and evaluation of EGFR status, particularly withregard to aberrant expression of EGFR, may be utilized to diagnose,evaluate and monitor patient samples including those known to have orsuspected of having cancer, a precancerous condition, a conditionrelated to hyperproliferative cell growth or from a tumor sample. Theassessment and evaluation of EGFR status is also useful in determiningthe suitability of a patient for a clinical trial of a drug or for theadministration of a particular chemotherapeutic agent or specificbinding member, particularly an antibody, of the present invention,including combinations thereof, versus a different agent or bindingmember. This type of diagnostic monitoring and assessment is already inpractice utilizing antibodies against the HER2 protein in breast cancer(Hercep Test, Dako Corporation), where the assay is also used toevaluate patients for antibody therapy using Herceptin. In vivoapplications include imaging of tumors or assessing cancer status ofindividuals, including radioimaging.

As suggested previously, the diagnostic method of the present inventioncomprises examining a cellular sample or medium by means of an assayincluding an effective amount of an antagonist to an EGFR/protein, suchas an anti-EGFR antibody, preferably an affinity-purified polyclonalantibody, and more preferably a mAb. In addition, it is preferable forthe anti-EGFR antibody molecules used herein be in the form of Fab,Fab′, F (ab′)₂ or F (v) portions or whole antibody molecules. Aspreviously discussed, patients capable of benefiting from this methodinclude those suffering from cancer, a pre-cancerous lesion, a viralinfection, pathologies involving or resulting from hyperproliferativecell growth or other like pathological derangement. Methods forisolating EGFR and inducing anti-EGFR antibodies and for determining andoptimizing the ability of anti-EGFR antibodies to assist in theexamination of the target cells are all well-known in the art.

Preferably, the anti-EGFR antibody used in the diagnostic methods ofthis invention is an affinity purified polyclonal antibody. Morepreferably, the antibody is a monoclonal antibody (mAb). In addition,the anti-EGFR antibody molecules used herein can be in the form of Fab,Fab′, F (ab′)₂ or F (v) portions of whole antibody molecules.

As described in detail above, antibody (ies) to the EGFR can be producedand isolated by standard methods including the well known hybridomatechniques. For convenience, the antibody (ies) to the EGFR will bereferred to herein as Ab_(i) and antibody (ies) raised in anotherspecies as Ab₂.

The presence of EGFR in cells can be ascertained by the usual in vitroor in vivo immunological procedures applicable to such determinations. Anumber of useful procedures are known. Three such procedures which areespecially useful utilize either the EGFR labeled with a detectablelabel, antibody Ab, labeled with a detectable label, or antibody Ab2labeled with a detectable label. The procedures may be summarized by thefollowing equations wherein the asterisk indicates that the particle islabeled, and “R” stands for the EGFR:

R*+Ab ₁ =R*Ab ₁,   A.

R+Ab*=RAb ₁*   B.

R+Ab ₁ +Ab ₂ *=RAb ₁ Ab ₂*   C.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the“sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known such as the “doubleantibody,” or “DASP” procedure.

In each instance above, the EGFR forms complexes with one or moreantibody (ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. For purposes of this description and claims, Ab₁ willbe referred to as a primary or anti-EGFR antibody, and Ab₂ will bereferred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine, auramine,Texas Red, AMCA blue and Lucifer Yellow. A particular detecting materialis anti-rabbit antibody prepared in goats and conjugated withfluorescein through an isothiocyanate.

The EGFR or its binding partner (s) such as the present specific bindingmember, can also be labeled with a radioactive element or with anenzyme. The radioactive label can be detected by any of the currentlyavailable counting procedures. The preferred isotope may be selectedfrom ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²¹I, ¹²⁴I,¹³¹I, ¹¹¹In, ²¹¹At, ¹⁹⁸Au, ⁶⁷Cu, ²²⁵Ac, ²¹³Bi, ⁹⁹Tc and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system that may be advantageously utilized inaccordance with the present invention, is known as a receptor assay. Ina receptor assay, the material to be assayed such as the specificbinding member, is appropriately labeled and then certain cellular testcolonies are inoculated with a quantity of both the labeled andunlabeled material after which binding studies are conducted todetermine the extent to which the labeled material binds to the cellreceptors. In this way, differences in affinity between materials can beascertained.

Accordingly, a purified quantity of the specific binding member may beradiolabeled and combined, for example, with antibodies or otherinhibitors thereto, after which binding studies would be carried out.Solutions would then be prepared that contain various quantities oflabeled and unlabeled uncombined specific binding member, and cellsamples would then be inoculated and thereafter incubated. The resultingcell monolayers are then washed, solubilized and then counted in a gammacounter for a length of time sufficient to yield a standard error of<5%. These data are then subjected to Scatchard analysis after whichobservations and conclusions regarding material activity can be drawn.While the foregoing is exemplary, it illustrates the manner in which areceptor assay may be performed and utilized, in the instance where thecellular binding ability of the assayed material may serve as adistinguishing characteristic.

An assay useful and contemplated in accordance with the presentinvention is known as a “cis/trans” assay. Briefly, this assay employstwo genetic constructs, one of which is typically a plasmid thatcontinually expresses a particular receptor of interest when transfectedinto an appropriate cell line, and the second of which is a plasmid thatexpresses a reporter such as luciferase, under the control of areceptor/ligand complex. Thus, for example, if it is desired to evaluatea compound as a ligand for a particular receptor, one of the plasmidswould be a construct that results in expression of the receptor in thechosen cell line, while the second plasmid would possess a promoterlinked to the luciferase gene in which the response element to theparticular receptor is inserted. If the compound under test is anagonist for the receptor, the ligand will complex with the receptor, andthe resulting complex will bind the response element and initiatetranscription of the luciferase gene. The resulting chemiluminescence isthen measured photometrically, and dose response curves are obtained andcompared to those of known ligands. The foregoing protocol is describedin detail in U.S. Pat. No. 4,981,784 and PCT International PublicationNo. WO 88/03168, for which purpose the artisan is referred.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of aberrant expression of EGFR, including but notlimited to overexpressed EGFR, amplified EGFR and/or an EGFR mutation,in suspected target cells. In accordance with the testing techniquesdiscussed above, one class of such kits will contain at least thelabeled EGFR or its binding partner, for instance an antibody specificthereto, and directions, of course, depending upon the method selected,e.g., “competitive,” “sandwich,” “DASP” and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for aberrant expression orpost-translational modification of EGFR, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of thepresent specific binding member or a specific binding partner thereto,to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of the specific binding member as described above (ora binding partner) generally bound to a solid phase to form animmunosorbent, or in the alternative, bound to a suitable tag, or pluralsuch end products, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g., “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

(a) a labeled component which has been obtained by coupling the specificbinding member to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

-   -   (i) a ligand capable of binding with the labeled component (a);    -   (ii) a ligand capable of binding with a binding partner of the        labeled component (a);    -   (iii) a ligand capable of binding with at least one of the        component (s) to be determined; and    -   (iv) a ligand capable of binding with at least one of the        binding partners of at least one of the component (s) to be        determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the EGFR, the specific binding member, and a specificbinding partner thereto.

In accordance with the above, an assay system for screening potentialdrugs effective to modulate the activity of the EGFR, the aberrantexpression or post-translational modification of the EGFR, and/or theactivity or binding of the specific binding member may be prepared. Thereceptor or the binding member may be introduced into a test system, andthe prospective drug may also be introduced into the resulting cellculture, and the culture thereafter examined to observe any changes inthe S-phase activity of the cells, due either to the addition of theprospective drug alone, or due to the effect of added quantities of theknown agent (s).

Nucleic Acids

The present invention further provides an isolated nucleic acid encodinga specific binding member of the present invention. Nucleic acidincludes DNA and RNA. In a preferred aspect, the present inventionprovides a nucleic acid which codes for a polypeptide of the inventionas defined above, including a polypeptide as set out as the CDR residuesof the VH and VL chains of the inventive antibodies.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell whichcomprises one or more constructs as above. A nucleic acid encoding anyspecific binding member as provided itself forms an aspect of thepresent invention, as does a method of production of the specificbinding member which method comprises expression from encoding nucleicacid therefore. Expression may conveniently be achieved by culturingunder appropriate conditions recombinant host cells containing thenucleic acid. Following production by expression a specific bindingmember may be isolated and/or purified using any suitable technique,then used as appropriate.

Specific binding members and encoding nucleic acid molecules and vectorsaccording to the present invention may be provided isolated and/orpurified, e.g. from their natural environment, in substantially pure orhomogeneous form, or, in the case of nucleic acid, free or substantiallyfree of nucleic acid or genes origin other than the sequence encoding apolypeptide with the required function. Nucleic acid according to thepresent invention may comprise DNA or RNA and may be wholly or partiallysynthetic.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Pluckthun, A. Bio/Technology 9:545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of a specific binding member, see forrecent reviews, for example Raff, M. E. (1993) Curr. Opinion Biotech.4:573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6:553-560.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.The disclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference.

Thus, a further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. A still further aspectprovides a method comprising introducing such nucleic acid into a hostcell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences which promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method which comprises using aconstruct as stated above in an expression system in order to express aspecific binding member or polypeptide as above.

As stated above, the present invention also relates to a recombinant DNAmolecule or cloned gene, or a degenerate variant thereof, which encodesa specific binding member, particularly antibody or a fragment thereof,that possesses an amino acid sequence set forth in SEQ ID NOS:2 and 4;129 and 134; 22 and 27; 32 and 37; and/or 42 and 47, preferably anucleic acid molecule, in particular a recombinant DNA molecule orcloned gene, encoding the binding member or antibody has a nucleotidesequence or is complementary to a DNA sequence encoding one of suchsequences.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAs, e.g., the numerous derivatives of phage X, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2u plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, YB/20, NSO, SP2/0, R1.1, B-W and L-M cells, AfricanGreen Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10),insect cells (e.g., Sf9), and human cells and plant cells in tissueculture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

It is further intended that specific binding member analogs may beprepared from nucleotide sequences of the protein complex/subunitderived within the scope of the present invention. Analogs, such asfragments, may be produced, for example, by pepsin digestion of specificbinding member material. Other analogs, such as muteins, can be producedby standard site-directed mutagenesis of specific binding member codingsequences. Analogs exhibiting “specific binding member activity” such assmall molecules, whether functioning as promoters or inhibitors, may beidentified by known in vivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding a specific binding membercan be prepared synthetically rather than cloned. The DNA sequence canbe designed with the appropriate codons for the specific binding memberamino acid sequence. In general, one will select preferred codons forthe intended host if the sequence will be used for expression. Thecomplete sequence is assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al.,Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express specific binding member analogs or “muteins”.Alternatively, DNA encoding muteins can be made by site-directedmutagenesis of native specific binding member genes or cDNAs, andmuteins can be made directly using conventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs withunnatural amino acids.

The present invention extends to the preparation of antisenseoligonucleotides and ribozymes that may be used to interfere with theexpression of the EGFR at the translational level. This approachutilizes antisense nucleic acid and ribozymes to block translation of aspecific mRNA, either by masking that mRNA with an antisense nucleicacid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (See Weintraub, 1990;Marcus-Sekura, 1988.). In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into producing cells. Antisense methods have been used to inhibitthe expression of many genes in vitro (Marcus-Sekura, 1988; Hambor etal., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988.).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type (Hasselhoff and Gerlach, 1988). Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”—typerecognize eleven-to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for EGFRs and theirligands.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLE 1 Generation and Isolation of Antibodies Cell Lines

For immunization and specificity analyses, several cell lines, native ortransfected with either the normal, wild-type or “wtEGFR” gene or theΔEGFR gene carrying the Δ2-7 deletion mutation were used: Murinefibroblast cell line NR6, NR6_(ΔEGFR) (transfected with ΔEGFR) andNR6_(wtEGFR) (transfected with wtEGFR), human glioblastoma cell lineU87MG (expressing low levels of endogenous wtEGFR), U87MG_(wtEGFR)(transfected with wtEGFR), U87MG_(ΔEGFR) (transfected with ΔEGFR), andhuman squamous cell carcinoma cell line A431 (expressing high levels ofwtEGFR).

For immunization and specificity analyses, several cell lines, native ortransfected with either the normal, wild-type or “wtEGFR” gene or theΔEGFR gene carrying the de2-7 or Δ2-7 deletion mutation were used:Murine fibroblast cell line NR6, NR6_(ΔEGFR) (transfected with ΔEGFR)and NR6_(wtEGFR) (transfected with wtEGFR), human glioblastoma cell lineU87MG (expressing low levels of endogenous wtEGFR), U87MG_(wtEGFR) or“U87MG.wtEGFR” (transfected with wtEGFR), U87MG_(ΔEGFR) or “U87MG.Δ2-7”(transfected with ΔEGFR), and human squamous cell carcinoma cell lineA431 (expressing high levels of wtEGFR). The NR6, NR6_(ΔEGFR), andNR6_(wtEGFR) cell lines were previously described (Batra et al. (1995)Epidermal Growth Factor Ligand-independent, Unregulated,Cell-Transforming Potential of a Naturally Occurring Human MutantEGFRvIII Gene. Cell Growth Differ. 6(10): 1251-1259). The NR6 cell linelacks normal endogenous EGFR. (Batra et al., 1995). U87MG cell lines andtransfections were described previously (Nishikawa et al. (1994) Amutant epidermal growth factor receptor common in human glioma confersenhanced tumorigenicity. Proc. Natl. Acad. Sci. U.S.A. 91, 7727-7731).

The U87MG astrocytoma cell line (Ponten, J. and Macintyre, E. H. (1968)Long term culture of normal and neoplastic human glia. Acta. Pathol.Microbiol. Scand. 74, 465-86) which endogenously expresses low levels ofthe wtEGFR, was infected with a retrovirus containing the de2-7 EGFR toproduce the U87MG.Δ2-7 cell line (Nishikawa et al., 1994). Thetransfected cell line U87MG.wtEGFR was produced as described in Naganeet al. (1996) Cancer Res. 56, 5079-5086. Whereas U87MG cells expressapproximately 1×10⁵ EGFR, U87MG.wtEGFR cells express approximately 1×10⁶EGFR, and thus mimic the situation seen with gene amplification. Themurine pro-B cell line BaF/3, which does not express any known EGFRrelated molecules, was also transfected with de2-7 EGFR. resulting inthe BaF/3. Δ2-7 cell line (Luwor et al. (2004) The tumor-specific de2-7epidermal growth factor receptor (EGFR) promotes cells survival andheterodimerizes with the wild-type EGFR, Oncogene 23: 6095-6104). Humansquamous carcinoma A431 cells were obtained from ATCC (Rockville, Md.).The epidermoid carcinoma cell line A431 has been described previously(Sato et al. (1987) Derivation and assay of biological effects ofmonoclonal antibodies to epidermal growth factor receptors. MethodsEnzymol. 146, 63-81).

All cell lines were cultured in DMEM/F-12 with G1utaMAX™ (LifeTechnologies, Inc., Melbourne, Australia and Grand Island, N.Y.)supplemented with 10% FCS (CSL, Melbourne, Australia); 2 mM glutamine(Sigma Chemical Co., St. Louis, Mo.), and penicillin/streptomycin (LifeTechnologies, Inc., Grand Island, N.Y.). In addition, the U87MG.Δ2-7 andU87MG.wtEGFR cell lines were maintained in 400 mg/ml of geneticin (LifeTechnologies, Inc., Melbourne, Victoria, Australia). Cell lines weregrown at 37° C. in a unmodified atmosphere of 5% CO₂.

Reagents

The de2-7 EGFR unique junctional peptide has the amino acid sequence:LEEKKGNYVVTDH (SEQ ID NO:13). Biotinylated unique junctional peptides(Biotin-LEEKKGNYVVTDH (SEQ ID NO:5) and LEEKKGNYVVTDH-Biotin (SEQ IDNO:6)) from de2-7 EGFR were synthesized by standard Fmoc chemistry andpurity (>96%) determined by reverse phase HPLC and mass spectralanalysis (Auspep, Melbourne, Australia).

Antibodies Used in Studies

In order to compare our findings with other reagents, additional mAbswere included in our studies. These reagents were mAb528 to the wtEGFR(Sato et al. (1983) Mol. Biol. Med. 1(5), 511-529) and DH8.3, which wasgenerated against a synthetic peptide spanning the junctional sequenceof the Δ2-7 EGFR deletion mutation. The DH8.3 antibody (IgG1), which isspecific for the de2-7 EGFR, has been described previously (Hills et al.(1995) Specific targeting of a mutant, activated EGF receptor found inglioblastoma using a monoclonal antibody. Int. J. Cancer. 63,537-43,1995) and was obtained following immunization of mice with theunique junctional peptide found in de2-7 EGFR (Hills et al., 1995).

The 528 antibody, which recognizes both de2-7 and wild-type EGFR, hasbeen described previously (Masui et al. (1984) Growth inhibition ofhuman tumor cells in athymic mice by anti-epidermal growth factorreceptor monoclonal antibodies. Cancer Res. 44, 1002-7) and was producedin the Biological Production Facility, Ludwig Institute for CancerResearch (Melbourne, Australia) using a hybridoma (ATCC HB-8509)obtained from the American Type Culture Collection (Rockville, Md.). Thepolyclonal antibody SC-03 is an affinity purified rabbit polyclonalantibody raised against a carboxy terminal peptide of the EGFR (SantaCruz Biotechnology Inc.).

Antibody Generation

The murine fibroblast line NR6_(EGFR) was used as immunogen. Mousehybridomas were generated by immunizing BALB/c mice five timessubcutaneously at 2- to 3-week intervals, with 5×10⁵-2×10⁶ cells inadjuvant. Complete Freund's adjuvant was used for the first injection.Thereafter, incomplete Freund's adjuvant (Difco™, Voigt GlobalDistribution, Lawrence, Kans.) was used. Spleen cells from immunizedmice were fused with mouse myeloma cell line SP2/0 (Shulman et al.(1978) Nature 276:269-270). Supernatants of newly generated clones werescreened in hemadsorption assays for reactivity with cell line NR6,NR6_(wtEGFR), and NR6_(ΔEGFR) and then analyzed by hemadsorption assayswith human glioblastoma cell lines U87MG, U87MG_(wtEGFR), andU87_(ΔEGFR). Selected hybridoma supernatants were subsequently tested bywestern blotting and further analyzed by immunohistochemistry. Newlygenerated mAbs showing the expected reactivity pattern were purified.

Five hybridomas were established and three clones, 124 (IgG2a), 806(IgG2b) (deposited as ATCC Deposit Number PTA-3858 on Nov. 4, 2001), and1133 (IgG2a) were initially selected for further characterization basedon high titer (1:2500) with NR6_(ΔEGFR) and low background on NR6 andNR6_(wtEGFR) cells in the rosette hemagglutination assay. A fourthclone, 175 (IgG2a) was subsequently further characterized and isdiscussed separately in Example 23, below. A fifth clone, 585 (IgG2a)was also further characterized, as discussed in Example 25, below. In asubsequent hemagglutination analysis, these antibodies showed noreactivity (undiluted supernatant ≦10%) with the native humanglioblastoma cell line U87MG and U87MG_(wtEGFR), but were stronglyreactive with U87MG_(ΔEGFR); less reactivity was seen with A431. Bycontrast, in FACS analysis, 806 was unreactive with native U87MG andintensively stained U87MG_(ΔEGFR) and to a lesser degree U87MG_(wtEGFR)indicating binding of 806 to both, ΔEGFR and wtEGFR (see below).

In Western blot assays, mAb124, mAb806 and mAbl 133 were then analyzedfor reactivity with wtEGFR and ΔEGFR. Detergent lysates were extractedfrom NR6_(EGFR), U87MG_(EGFR) as well as from A431. All three mAbsshowed a similar reactivity pattern with cell lysates staining both thewtEGFR (170 kDa) and ΔEGFR protein (140 kDa). As a reference reagent,mAbR.I. known to be reactive with the wtEGFR (Waterfield et al. (1982)J. Cell Biochem. 20(2), 149-161) was used instead of mAb528, which isknown to be non-reactive in western blot analysis. mAbR.I. showedreactivity with wild-type and ΔEGFR. All three newly generated clonesshowed reactivity with ΔEGFR and less intense with wtEGFR. DH8.3 wassolely positive in the lysate of U87MG_(ΔEGFR) and NR6_(ΔEGFR).

The immunohistochemical analysis of clones 124, 806, and 1133 as well asmAb528 and mAbDH8.3 on xenograft tumors U87MG, U87MG_(ΔEGFR), and A431are shown in Table 1. All mAbs showed strong staining of xenograftU87MG_(ΔEGFR). Only mAb528 showed weak reactivity in the native U87MGxenograft. In A431 xenografts, mAb528 showed strong homogeneousreactivity. mAb124, mAb806, and mAbl 133 revealed reactivity with mostlythe basally located cells of the squamous cell carcinoma of A431 and didnot react with the upper cell layers or the keratinizing component.DH8.3 was negative in A431 xenografts.

TABLE 1 Immunohistochemical Analysis of Antibodies 528, DH8.3, and 124,806 and 1133 xenograft xenograft Antibody ΔU87MG_(ΔEGFR) xenograft A431U87MG(native) mAb528 pos. pos. pos. (focal staining) mAb124 pos. pos.(predominantly — basal cells) mAb806 pos. pos. (predominantly — basalcells) mAb1133 pos. pos. (predominantly — basal cells) DH8.3 pos. — —minor stromal staining due to detection of endogenous mouse antibodies.

Sequencing

The variable heavy (VH) and variable light (VL) chains of mAb806, mAb124and mAb1133 were sequenced, and their complementarity determiningregions (CDRs) identified, as follows:

mAb806

mAb806 VH chain: nucleic acid sequence (SEQ ID NO:1) and amino acidsequence, with signal peptide (SEQ ID NO:2) are shown in FIGS.14A and14B, respectively (signal peptide underlined in FIG.14B).Complementarity determining regions CDR1, CDR2, and CDR3 (SEQ ID NOS:15, 16, and 17, respectively) are indicated by underlining in FIG.16.The mAb806 VH chain amino acid sequence without its signal peptide (SEQID NO:11) is shown in FIG. 16.

mAb806 VL chain: nucleic acid sequence (SEQ ID NO:3) and amino acidsequence, with signal peptide (SEQ ID NO:4) are shown in FIGS. 15A and15B, respectively (signal peptide underlined in FIG. 15B).Complementarity determining regions CDR1, CDR2, and CDR3 (SEQ ID NOS:18, 19, and 20, respectively) are indicated by underlining in FIG. 17.The mAb806 VL chain amino acid sequence without its signal peptide (SEQID NO:12) is shown in FIG. 17.

mAb124

mAb124 VH chain: nucleic acid (SEQ ID NO:21) and amino acid (SEQ IDNO:22) sequences are shown in FIGS. 51A and 51B, respectively.Complementarity determining regions CDR1, CDR2, and CDR3 (SEQ ID NOS:23, 24, and 25, respectively) are indicated by underlining.

mAb124 VL chain: nucleic acid (SEQ ID NO:26) and amino acid (SEQ IDNO:27) sequences are shown in FIGS. 51C and 51D, respectively.Complementarity determining regions CDR1, CDR2, and CDR3 (SEQ ID NOS:28, 29, and 30, respectively) are indicated by underlining.

mAb1133

mAb1113 VH chain: nucleic acid (SEQ ID NO:31) and amino acid (SEQ IDNO:32) sequences are shown in FIGS. 52A and 52B, respectively.Complementarity determining regions CDR1, CDR2, and CDR3 (SEQ ID NOS:33, 34, and 35, respectively) are indicated by underlining.

mAb1133 VL chain: nucleic acid (SEQ ID NO:36) and amino acid (SEQ IDNO:37) sequences are shown in FIGS. 52C and 52D, respectively.Complementarity determining regions CDR1, CDR2, and CDR3 (SEQ ID NOS:38, 39, and 40, respectively) are indicated by underlining.

EXAMPLE 2 Binding of Antibodies to Cell Lines by FACS

mAb806 was initially selected for further characterization, as set forthherein and in the following Examples. mAb 124 and mAb 1133 were alsoselected for further characterization, as discussed in Example 24 below,and found to have properties corresponding to the unique properties ofmAb806 discussed herein.

In order to determine the specificity of mAb806, its binding to U87MG,U87MG.Δ2-7 and U87MG.wtEGFR cells was analyzed by flow activated cellsorting (FACS). Briefly, cells were labelled with the relevant antibody(10 μg/ml) followed by fluorescein-conjugated goat anti-mouse IgG (1:100dilution; Calbiochem San Diego, CA, USA; Becton-Dickinson PharMingen,San Diego, CA, US) as described previously (Nishikawa et al., 1994).FACS data was obtained on a Coulter Epics Elite ESP by observing aminimum of 5,000 events and analyzed using EXPO (version 2) for Windows.An irrelevant IgG2b was included as an isotype control for mAb806 andthe 528 antibody was included as it recognizes both the de2-7 andwtEGFR.

Only the 528 antibody was able to stain the parental U87MG cell line(FIG.1) consistent with previous reports demonstrating that these cellsexpress the wtEGFR (Nishikawa et al, 1994). mAb806 and DH8.3 had bindinglevels similar to the control antibody, clearly demonstrating that theyare unable to bind the wild-type receptor (FIG.1). Binding of theisotype control antibody to U87MG.Δ2-7 and U87MG.wtEGFR cells wassimilar as that observed for the U87MG cells.

mAb806 stained U87MG.Δ2-7 and U87MG.wtEGFR cells, indicating that mAb806specifically recognizes the de2-7 EGFR and amplified EGFR (FIG.1). DH8.3antibody stained U87MG.Δ2-7 cells, confirming that DH8.3 antibodyspecifically recognizes the de2-7 EGFR (FIG.1). As expected, the 528antibody stained both the U87MG.Δ2-7 and U87MG.wtEGFR cell lines (FIG.1). As expected, the 528 antibody stained U87MG.Δ2-7 with a higherintensity than the parental cell as it binds both the de2-7 andwild-type receptors that are co-expressed in these cells (FIG. 1).Similar results were obtained using a protein A mixed hemadsorptionwhich detects surface bound IgG by appearance of Protein A coated withhuman red blood cells (group O) to target cells. Monoclonal antibody 806was reactive with U87MG.Δ2-7 cells but showed no significant reactivity(undiluted supernatant less than 10%) with U87MG expressing wild-typeEGFR. Importantly, mAb806 also bound the BaF/3.Δ2-7 cell line,demonstrating that the co-expression of wtEGFR is not a requirement formAb806 reactivity (FIG. 1).

EXAMPLE 3 Binding of Antibodies in Assays

To further characterize the specificity of mAb806 and the DH8.3antibody, their binding was examined by ELISA. Two types of ELISA wereused to determine the specificity of the antibodies. In the first assay,plates were coated with sEGFR (10 μg/ml in 0.1 M carbonate buffer pH9.2) for 2 h and then blocked with 2% human serum albumin (HSA) in PBS.sEGFR is the recombinant extracellular domain (amino acids 1-621) of thewild-type EGFR), and was produced as previously described (Domagala etal. (2000) Stoichiometry, kinetic and binding analysis of theinteraction between Epidermal Growth Factor (EGF) and the ExtracellularDomain of the EGF receptor. Growth Factors. 18, 11-29). Antibodies wereadded to wells in triplicate at increasing concentration in 2% HSA inphosphate-buffered saline (PBS). Bound antibody was detected byhorseradish peroxidase conjugated sheep anti-mouse IgG (Silenus,Melbourne, Australia) using ABTS (Sigma, Sydney, Australia) as asubstrate and the absorbance measured at 405 nm.

Both mAb806 and the 528 antibody displayed dose-dependent and saturatingbinding curves to immobilized wild-type sEGFR (FIG. 2A). As the uniquejunctional peptide found in the de2-7 EGFR is not contained within thesEGFR, mAb806 must be binding to an epitope located within the wild-typeEGFR sequence. The binding of the 528 antibody was lower than thatobserved for mAb806, probably because it recognizes a conformationaldeterminant. As expected, the DH8.3 antibody did not bind the wild-typesEGFR even at concentrations up to 10 μg/ml (FIG. 2A). Although sEGFR insolution inhibited the binding of the 528 antibody to immobilized sEGFRin a dose-dependent fashion, it was unable to inhibit the binding ofmAb806 (FIG.2B). This suggests that mAb806 can only bind wild-type EGFRonce immobilized on ELISA plates, a process that may induceconformational changes. Similar results were observed using a BlAcorewhereby mAb806 bound immobilized sEGFR but immobilized mAb806 was notable to bind sEGFR in solution (FIG. 2C).

Following denaturation by heating for 10 min at 95° C., sEGFR insolution was able to inhibit the binding of mAb806 to immobilized sEGFR(FIG. 2C), confirming that mAb806 can bind the wild-type EGFR undercertain conditions. Interestingly, the denatured sEGFR was unable toinhibit the binding of the 528 antibody (FIG. 2C), demonstrating thatthis antibody recognizes a conformational epitope. The DH8.3 antibodyexhibited dose-dependent and saturable binding to the unique de2-7 EGFRpeptide (FIG.2D). Neither mAb806 or the 528 antibody bound to thepeptide, even at concentrations higher than those used to obtainsaturation binding of DH8.3, further indicating mAb806 does notrecognize an epitope determinant within this peptide.

In the second assay, the biotinylated de2-7 specific peptide (BiotinLEEKKGNYVVTDH (SEQ ID NO:5)) was bound to ELISA plates precoated withstreptavidin (Pierce, Rockford, Illinois). Antibodies were bound anddetected as in the first assay. Neither mAb806 nor the 528 antibodybound to the peptide, even at concentrations higher than those used toobtain saturation binding of DH8.3, further indicating that mAb806 doesnot recognize an epitope determinant within this peptide.

To further demonstrate that mAb806 recognizes an epitope distinct fromthe junction peptide, additional experiments were performed. C-terminalbiotinylated de2-7 peptide (LEEKKGNYVVTDH-Biotin (SEQ ID NO:6)) wasutilized in studies with mAb806 and mAbL8A4, generated against the de2-7peptide (Reist et al. (1995) Cancer Res. 55(19), 4375-4382; Foulon etal. (2000) Cancer Res. 60(16), 4453-4460).

Reagents Used in Peptide Studies

-   Junction Peptide: LEEKKGNYVVTDH-OH (Biosource, Camarillo, Calif.);-   Peptide C: LEEKKGNYVVTDH(K-Biot)-OH (Biosource, Camarillo, Calif.);-   sEGFR: CHO-cell-derived recombinant soluble extracellular domain    (amino acids 1-621) of the wild-type EGFR (LICR Melbourne);-   mAb806: mouse monoclonal antibody, IgG_(2b) (LICR NYB);-   mAbL8A4: mouse monoclonal antibody, IgG₁ (Duke University);-   IgG₁ isotype control mAb;-   IgG_(2b) isotype control mAb.

Peptide C was immobilized on a Streptavidin microsensor chip at asurface density of 350RU (+/−30RU). Serial dilutions of mAbs were testedfor reactivity with the peptide. Blocking experiments usingnon-biotinylated peptide were performed to assess specificity.

mAbL8A4 showed strong reactivity with Peptide C even at low antibodyconcentrations (6.25 nM) (FIG.2E). mAb806 did not show detectablespecific reactivity with Peptide C up to antibody concentrations of 100nM (highest concentration tested) (FIGS. 2E and 2F). It was expectedthat mAbL8A4 would react with Peptide C because the peptide was used asthe immunogen in the generation of mAbL8A4. Addition of the JunctionPeptide (non-biotinylated, 50 μg/ml) completely blocks the reactivity ofmAbL8A4 with Peptide C, confirming the antibody's specificity for thejunction peptide epitope.

In a second set of BIAcore experiments, sEGFR was immobilized on a CMmicrosensor chip at a surface density of ˜4000 RU. Serial dilutions ofmAbs were tested for reactivity with sEGFR.

mAb806 was strongly reactive with denaturated sEGFR while mAbL8A4 didnot react with denaturated sEGFR. Reactivity of mAb806 with denaturatedsEGFR decreases with decreasing antibody concentrations. It was expectedthat mAbL8A4 does not react with sEGFR because mAbL8A4 was generatedusing the junction peptide as the immunogen and sEGFR does not containthe junction peptide.

Dot-blot immune stain experiments were also performed. Serial dilutionsof peptide were spotted in 0.5 μl onto a PVDF or nitrocellulosemembranes. Membranes were blocked with 2% BSA in PBS, and then probedwith 806, L8A4, DH8.3 and control antibodies. Antibodies L8A4 and DH8.3bound to peptide on the membranes (data not shown). mAb806 did not bindpeptide at concentrations where L8A4 clearly showed binding (data notshown). Control antibodies were also negative for peptide binding.

mAb806 bound to the wtEGFR in cell lysates following immunoblotting(results not shown). This is different from the results obtained withDH8.3 antibody, which reacted with de2-7 EGFR but not wtEGFR. Thus,mAb806 can recognize the wtEGFR following denaturation but not when thereceptor is in its natural state on the cell surface.

EXAMPLE 4 Scatchard Analysis

A Scatchard analysis using U87MG.Δ2-7 cells was performed followingcorrection for immunoreactivity in order to determine the relativeaffinity of each antibody. Antibodies were labelled with ¹²⁵I (Amrad,Melbourne, Australia) by the Chloramine T method and immunoreactivitydetermined by Lindmo assay (Lindmo et al. (1984) Determination of theimmunoreactive fraction of radiolabeled monoclonal antibodies by linearextrapolation to binding at infinite antigen excess. J. Immunol.Methods. 72, 77-89).

All binding assays were performed in 1% HSA/PBS on 1-2×10⁶ liveU87MG.Δ2-7 or A431 cells for 90 min at 4° C. with gentle rotation. A setconcentration of 10 ng/ml ¹²⁵I-labeled antibody was used in the presenceof increasing concentrations of the appropriate unlabeled antibody.Non-specific binding was determined in the presence of 10,000-foldexcess of unlabeled antibody. Neither ¹²⁵I-radiolabeled mAb806 or theDH8.3 antibody bound to parental U87MG cells. After the incubation wascompleted, cells were washed and counted for bound ¹²⁵I-labeled antibodyusing a COBRA II gamma counter (Packard Instrument Company, Meriden,Conn., USA).

Both mAb806 and the DH8.3 antibody retained high immunoreactivity wheniodinated and was typically greater than 90% for mAb806 and 45-50% forthe DH8.3 antibody. mAb806 had an affinity for the de2-7 EGFR receptorof 1.1 x 10⁹ M⁻¹ whereas the affinity of DH8.3 was some 10-fold lower at1.0×10⁸ M⁻¹. Neither iodinated antibody bound to U87MG parental cells.mAb806 recognized an average of 2.4×10⁵ binding sites per cell with theDH8.3 antibody binding an average of 5.2×10⁵ sites. Thus, there was notonly good agreement in receptor number between the antibodies, but alsowith a previous report showing 2.5×10⁵ de2-7 receptors per cell asmeasured by a different de2-7 EGFR specific antibody on the same cellline (Reist et al. (1997) Improved targeting of an anti-epidermal growthfactor receptor variant III monoclonal antibody in tumor xenograftsafter labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate. CancerRes. 57, 1510-5).

EXAMPLE 5 Internalization of Antibodies By U87MG.Δ2-7 Cells

The rate of antibody internalization following binding to a target cellinfluences both its tumor targeting properties and therapeutic options.Consequently, the inventors examined the internalization of mAb806 andthe DH8.3 antibody following binding to U87MG.Δ2-7 cells by FACS.U87MG.Δ2-7 cells were incubated with either mAb806 or the DH8.3 antibody(10 μg/ml) for 1 h in DMEM at 4° C. After washing, cells weretransferred to DMEM pre-warmed to 37° C. and aliquots taken at varioustime points following incubation at 37° C. Internalization was stoppedby immediately washing aliquots in ice-cold wash buffer (1% HSA/PBS). Atthe completion of the time course cells were stained by FACS asdescribed above. Percentage internalization was calculated by comparingsurface antibody staining at various time points to zero time using theformula: percent antibody internalized=(mean fluorescence attime_(x)−background fluorescence)/(mean fluorescence at time₀−backgroundfluorescence)×100. This method was validated in one assay using aniodinated antibody (mAb806) to measure internalization as previouslydescribed (Huang et al. (1997) The enhanced tumorigenic activity of amutant epidermal growth factor receptor common in human cancers ismediated by threshold levels of constitutive tyrosine phosphorylationand unattenuated signaling. J. Biol. Chem. 272, 2927-35). Differences ininternalization rate at different time points were compared usingStudent's t-test. Throughout this research, data were analyzed forsignificance by Student's t-test, except for the in vivo survivalassays, which were analyzed by Wilcoxon analysis.

Both antibodies showed relatively rapid internalization reachingsteady-state levels at 10 min for mAb806 and 30 min for DH8.3 (FIG.3).Internalization of DH8.3 was significantly higher both in terms of rate(80.5% of DH8.3 internalized at 10 min compared to 36.8% for mAb806,p<0.01) and total amount internalized at 60 min (93.5% versus 30.4%,p<0.001). mAb806 showed slightly lower levels of internalization at 30and 60 min compared to 20 min in all 4 assays performed (FIG.3). Thisresult was also confirmed using an internalization assay based oniodinated mAb806 (data not shown).

EXAMPLE 6 Electron Microscopy Analysis of Antibody Internalization

Given the above noted difference in internalization rates between theantibodies, a detailed analysis of antibody intracellular traffickingwas performed using electron microscopy.

U87MG.Δ2-7 cells were grown on gelatin coated chamber slides (Nunc,Naperville, Ill.) to 80% confluence and then washed with ice cold DMEM.Cells were then incubated with mAb806 or the DH8.3 antibody in DMEM for45 min at 4° C. After washing, cells were incubated for a further 30 minwith gold-conjugated (20 nm particles) anti-mouse IgG (BBlnternational,Cardiff, UK) at 4° C. Following a further wash, pre-warmed DMEM/10% PCSwas added to the cells, which were incubated at 37° C. for various timesfrom 1-60 min. Internalization of the antibody was stopped by ice-coldmedia and cells fixed with 2.5% glutaraldehyde in PBS/0.1% HSA and thenpost-fixed in 2.5% osmium tetroxide. After dehydration through a gradedseries of acetone, samples were embedded in Epon/Araldite resin, cut asultra-thin sections with a Reichert Ultracut-S microtome (Leica) andcollected on nickel grids. The sections were stained with uranyl acetateand lead citrate before being viewed on a Philips CM12 transmissionelectron microscope at 80 kV. Statistical analysis of gold grainscontained within coated pits was performed using a Chi-square test.

While the DH8.3 antibody was internalized predominantly via coated-pits,mAb806 appeared to be internalized by macropinocytosis (FIG. 19). Infact, a detailed analysis of 32 coated pits formed in cells incubatedwith mAb806 revealed that none of them contained antibody. In contrast,around 20% of all coated-pits from cells incubated with DH8.3 werepositive for antibody, with a number containing multiple gold grains. Astatistical analysis of the total number of gold grains contained withincoated-pits found that the difference was highly significant (p<0.01).After 20-30 min both antibodies could be seen in structures thatmorphologically resemble lysosomes (FIG. 19C). The presence of cellulardebris within these structures was also consistent with their lysosomenature.

EXAMPLE 7 Biodistribution of Antibodies in Tumor Bearing Nude Mice

The biodistribution of mAb806 and the DH8.3 antibody was compared innude mice containing U87MG xenografts on one side and U87MG.Δ2-7xenografts on the other. A relatively short time period was chosen forthis study as a previous report demonstrated that the DH8.3 antibodyshows peak levels of tumor targeting between 4-24 h (Hills et al. (1995)Specific targeting of a mutant, activated EGF receptor found inglioblastoma using a monoclonal antibody. Int. J. Cancer. 63, 537-43).

Tumor xenografts were established in nude BALB/c mice by s.c. injectionof 3×10⁶ U87MG, U87MG.Δ2-7 or A431 cells. de2-7 EGFR expression inU87MG.Δ2-7 xenografts remained stable throughout the period ofbiodistribution as measured by immunohistochemistry at various timepoints (data not shown). A431 cells retained their mAb806 reactivitywhen grown as tumor xenografts as determined by immunohistochemistry.U87MG or A431 cells were injected on one side 7-10 days beforeU87MG.Δ2-7 cells were injected on the other side because of the fastergrowth rate observed for de2-7 EGFR expressing xenografts. Antibodieswere radiolabeled and assessed for immunoreactivity as described aboveand were injected into mice by the retro-orbital route when tumors were100-200 mg in weight. Each mouse received two different antibodies (2 μgper antibody): 2 μCi of ¹²⁵I-labeled mAb806 and 2 μCi of ¹³¹I labelledDH8.3 or 528. Unless indicated, groups of 5 mice were sacrificed atvarious time points post-injection and blood obtained by cardiacpuncture. The tumors, liver, spleen, kidneys and lungs were obtained bydissection. All tissues were weighed and assayed for ¹²⁵I and ¹³¹Iactivity using a dual-channel counting Window. Data was expressed foreach antibody as % ID/g tumor determined by comparison to injected dosestandards or converted into tumor to blood/liver ratios (i.e. % ID/gtumor divided by % ID/g blood or liver). Differences between groups wereanalyzed by Student's t-test. After injection of radiolabeled mAb806,some tumors were fixed in formalin, embedded in paraffin, cut into 5, umsections and then exposed to X-ray film (AGFA, Mortsel, Belgium) todetermine antibody localization by autoradiography.

In terms of % ID/g tumor, mAb806 reached its peak level in U87MG.Δ2-7xenografts of 18.6% m/g tumor at 8 h (FIG. 4A), considerably higher thanany other tissue except blood. While DH8.3 also showed peak tumor levelsat 8 h, the level was a statistically (p <0.001) lower 8.8% m/g tumorcompared to mAb806 (FIG. 4B). Levels of both antibodies slowly declinedat 24 and 48 h. Autoradiography of U87MG.Δ2-7 xenograft tissue sectionscollected 8 hr after injection with ¹²⁵I-labeled mAb806 alone, clearlyillustrates localization of antibody to viable tumor (FIG. 20). Neitherantibody showed specific targeting of U87MG parental xenografts (FIGS.4A and 4B). With regards to tumor to blood/liver ratios, mAb806 showedthe highest ratio at 24 h for both blood (ratio of 1.3) and liver (ratioof 6.1) (FIGS. 5A and 5B). The DH8.3 antibody had its highest ratio inblood at 8 h (ratio of 0.38) and at 24 h in liver (ratio of 1.5) (FIGS.5A and 5B), both of which are considerably lower than the valuesobtained for mAb806.

As described above, levels of mAb806 in the tumor peaked at 8 hours.While this peak is relatively early compared to many tumor-targetingantibodies, it is completely consistent with other studies using de2-7EGFR specific antibodies which all show peaks at 4-24 hourspost-injection when using a similar dose of antibody (Hills et al.,1995; Reist et al., 1997; Reist et al. (1996) Radioiodination ofinternalizing monoclonal antibodies using N-succinimidyl5-iodo-3-pyridinecarboxylate. Cancer Res. 56, 4970-7). Indeed, unlikethe earlier reports, the 8 h time point was included on the assumptionthat antibody targeting would peak rapidly. The % ID/g tumor seen withmAb806 was similar to that reported for other de2-7 EGFR specificantibodies when using standard iodination techniques (Hills et al.,1995; Huang et al., 1997; Reist et al. (1995) Tumor-specificanti-epidermal growth factor receptor variant III monoclonal antibodies:use of the tyramine-cellobiose radioiodination method enhances cellularretention and uptake in tumor xenografts. Cancer Res. 55, 4375-82).

The reason for the early peak is probably two-fold. Firstly, tumorsexpressing the de2-7 EGFR, including the transfected U87MG cells, growextremely rapidly as tumor xenografts. Thus, even during the relativelyshort period of time used in these biodistribution studies, the tumorsize increases to such an extent (5-10 fold increase in mass over 4days) that the % ID/g tumor is reduced compared with slow growingtumors. Secondly, while internalization of mAb806 was relatively slowcompared to DH8.3, it is still rapid with respect to many other tumorantibody/antigen systems. Internalized antibodies undergo rapidproteolysis with the degradation products being excreted from the cell(Press et al. (1990) Inhibition of catabolism of radiolabeled antibodiesby tumor cells using lysosomotropic amines and carboxylic ionophores.Cancer Res. 50, 1243-50). This process of internalization, degradationand excretion reduces the amount of iodinated antibody retained withinthe cell. Consequently, internalizing antibodies display lower levels oftargeting than their non-internalizing counterparts. The electronmicroscopy data reported herein demonstrates that internalized mAb806 israpidly transported to lysosomes where rapid degradation presumablyoccurs. This observation is consistent with the swift expulsion ofiodine from the cell.

The previously described L8A4 monoclonal antibody directed to the uniquejunctional peptide found in the de2-7 EGFR, behaves in a similar fashionto mAb806 (Reist et al. (1997) In vitro and in vivo behavior ofradiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison withits murine parent. Nucl. Med. Biol. 24, 639-47). Using U87MG cellstransfected with the de2-7 EGFR, this antibody had a similarinternalization rate (35% at 1 hour compared to 30% at 1 hour formAb806) and displayed comparable in vivo targeting when using 3T3fibroblasts transfected with de2-7 EGFR (peak of 24% ID/g tumor at 24hours compared to 18% ID/g tumor at 8 hours for mAb806) (Reist et al.(1997) Improved targeting of an anti-epidermal growth factor receptorvariant III monoclonal antibody in tumor xenografts after labeling usingN-succinimidyl 5-iodo-3-pyridinecarboxylate. Cancer Res. 57, 1510-5).

Interestingly, in vivo retention of this antibody in tumor xenograftswas enhanced when labeled with N-succinimidyl 5-iodo-3-pyridinecarboxylate (Reist et al., 1997). This labeled prosthetic group ispositively charged at lysosmal pH and thus has enhanced cellularretention (Reist et al. (1996) Radioiodination of internalizingmonoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate.Cancer Res. 56, 4970-7). Enhanced retention is potentially useful whenconsidering an antibody for radioimmunotherapy and this method could beused to improve retention of iodinated mAb806 or its fragments.

EXAMPLE 8 Binding of mAb806 to Cells Containing Amplified EGFR

To examine if mAb806 could recognize the EGFR expressed in cellscontaining an amplified receptor gene, its binding to A431 cells wasanalyzed. As described previously, A431 cells are human squamouscarcinoma cells and express high levels of wtEGFR. Low, but highlyreproducible, binding of mAb806 to A431 cells was observed by FACSanalysis (FIG. 6). The DH8.3 antibody did not bind A431 cells,indicating that the binding of mAb806 was not the result of low levelde2-7 EGFR expression (FIG. 6). As expected, the anti-EGFR 528 antibodyshowed strong staining of A431 cells (FIG. 6). Given this result,binding of mAb806 to A431 was characterized by Scatchard analysis. Whilethe binding of iodinated mAb806 was comparatively low, it was possibleto get consistent data for Scatchard. The average of three suchexperiments gave a value for affinity of 9.5×10⁷ M⁻¹ with 2.4×10⁵receptors per cell. Thus, the affinity for this receptor was some10-fold lower than the affinity for the de2-7 EGFR. Furthermore, mAb806appears to only recognize a small portion of EGFR found on the surfaceof A431 cells. The 528 antibody measured approximately 2×10⁶ receptorsper cell which is in agreement with numerous other studies (Santon etal. (1986) Effects of epidermal growth factor receptor concentration ontumorigenicity of A431 cells in nude mice. Cancer Res. 46, 4701-5).

To ensure that these results were not simply restricted to the A431 cellline, mAb806 reactivity was examined in 2 other cells lines exhibitingamplification of the EGFR gene. Both the FINS head and neck cell line(Kwok T T and Sutherland R M (1991) Differences in EGF relatedradiosensitisation of human squamous carcinoma cells with high and lownumbers of EGF receptors. Br. J. Cancer. 64, 251-4) and the MDA-468breast cancer cell line (Filmus et al. (1985) MDA-468, a human breastcancer cell line with a high number of epidermal growth factor (EGF)receptors, has an amplified EGF receptor gene and is growth inhibited byEGF. Biochem. Biophys. Res. Commun. 128, 898-905) have been reported tocontain multiple copies of the EGFR gene. Consistent with these reports,the 528 antibody displayed intense staining of both cell lines (FIG.21). As with the A431 cell line, the mAb806 clearly stained both celllines but at a lower level than that observed with the 528 antibody(FIG. 21). Thus, mAb806 binding is not simply restricted to A431 cellsbut appears to be a general observation for cells containingamplification of the EGFR gene.

Recognition of the wild-type sEGFR by mAb806 clearly requires somedenaturation of the receptor in order to expose the epitope. The extentof denaturation required is only slight as even absorption of thewild-type sEGFR on to a plastic surface induced robust binding of mAb806in ELISA assays. As mAb806 only bound approximately 10% of the EGFR onthe surface of A431 cells, it is tempting to speculate that this subsetof receptors may have an altered conformation similar to that induced bythe de2-7 EGFR truncation. Indeed, the extremely high expression of theEGFR mediated by gene amplification in A431 cells may cause somereceptors to be incorrectly processed leading to altered conformation.Interestingly, semi-quantitative immunoblotting of A431 cell lysateswith mAb806 showed that it could recognize most of the A431 EGFreceptors following SDS-PAGE and western transfer. This result furthersupports the argument that mAb806 is binding to a subset of receptors onthe surface of A431 cells that have an altered conformation. Theseobservations in A431 cells are consistent with the immunohistochemistrydata demonstrating that mAb806 binds gliomas containing amplification ofthe EGFR gene. As mAb806 binding was completely negative on parentalU87MG cells it would appear this phenomenon may be restricted to cellscontaining amplified EGFR although the level of “denatured” receptor onthe surface of U87MG cells may be below the level of detection. However,this would seem unlikely as iodinated mAb806 did not bind to U87MG cellpellets containing up to 1×10⁷ cells.

EXAMPLE 9 In Vivo Targeting of A431 Cells By mAb806

A second biodistribution study was performed with mAb806 to determine ifit could target A431 tumor xenografts. The study was conducted over alonger time course in order obtain more information regarding thetargeting of U87MG.Δ2-7 xenografts by mAb806, which were included in allmice as a positive control. In addition, the anti-EGFR 528 antibody wasincluded as a positive control for the A431 xenografts, since a previousstudy demonstrated low but significant targeting of this antibody toA431 cells grown in nude mice (Masui et al. (1984) Growth inhibition ofhuman tumor cells in athymic mice by anti-epidermal growth factorreceptor monoclonal antibodies. Cancer Res. 44, 1002-7).

During the first 48 h, mAb806 displayed almost identical targetingproperties as those observed in the initial experiments (FIG.7A comparedwith FIG.4A). In terms of % ID/g tumor, levels of mAb806 in U87MG.Δ2-7xenografts slowly declined after 24 h but always remained higher thanlevels detected in normal tissue. Uptake in the A431 xenografts wascomparatively low, however there was a small increase in % ID/g tumorduring the first 24 h not observed in normal tissues such as liver,spleen, kidney and lung (FIG.7A). Uptake of the 528 antibody was verylow in both xenografts when expressed as % ID/g tumor (FIG.7B) partiallydue to the faster clearance of this antibody from the blood.Autoradiography of A431 xenograft tissue sections collected 24 hr afterinjection with ¹²⁵I-labeled mAb806 alone, clearly illustrateslocalization of antibody to viable tumor around the periphery of thetumor and not central areas of necrosis (FIG. 23). In terms of tumor toblood ratio mAb806 peaked at 72 h for U87MG.Δ2-7 xenografts and 100 hfor A431 xenografts (FIGS.8A, B). While the tumor to blood ratio formAb806 never surpassed 1.0 with respect to the A431 tumor, it didincrease throughout the entire time course (FIG. 8B) and was higher thanall other tissues examined (data not shown) indicating low levels oftargeting.

The tumor to blood ratio for the 528 antibody showed a similar profileto mAb806 although higher levels were noted in the A431 xenografts(FIGS. 8A, B). mAb806 had a peak tumor to liver ratio in U87MG.Δ2-7xenografts of 7.6 at 72 h, clearly demonstrating preferential uptake inthese tumors compared to normal tissue (FIG.8C). Other tumor to organratios for mAb806 were similar to those observed in the liver (data notshown). The peak tumor to liver ratio for mAb806 in A431 xenografts was2.0 at 100 h, again indicating a slight preferential uptake in tumorcompared with normal tissue (FIG. 8D).

EXAMPLE 10 Therapy Studies

The effects of mAb806 were assessed in two xenograft models of disease—apreventative model and an established tumor model.

Xenograft Models

Consistent with previous reports (Nishikawa et al., Proc. Natl. Acad.Sci. U.S.A., 91(16), 7727-7731), U87MG cells transfected with de2-7 EGFRgrew more rapidly than parental cells and U87MG cells transfected withthe wtEGFR. Therefore, it was not possible to grow both cell types inthe same mice.

Tumor cells (3×10⁶) in 100 ml of PBS were inoculated subcutaneously intoboth flanks of 4-6 week old female nude mice (Animal Research Centre,Western Australia, Australia). Therapeutic efficacy of mAb806 wasinvestigated in both preventative and established tumor models. In thepreventative model, 5 mice with two xenografts each were treatedintraperitoneally with either 1 or 0.1 mg of mAb806 or vehicle (PBS)starting the day before tumor cell inoculation. Treatment was continuedfor a total of 6 doses, 3 times per week for 2 weeks. In the establishedmodel, treatment was started when tumors had reached a mean volume of65±6.42 mm³ (U87MG.Δ2-7), 84±9.07 mm3 (U87MG), 73±7.5 mm³ (U87MG.wtEGFR)or 201±19.09 mm³ (A431 tumors). Tumor volume in mm³ was determined usingthe formula (length×width²)/2, where length was the longest axis andwidth the measurement at right angles to the length (Clark et al. (2000)Therapeutic efficacy of anti-Lewis (y) humanized 3S 193radioimmunotherapy in a breast cancer model: enhanced activity whencombined with Taxol chemotherapy. Clin. Cancer Res. 6, 3621-3628). Datawas expressed as mean tumor volume±S.E. for each treatment group.Statistical analysis was performed at given time points using Student'st-test. Animals were euthanized when the xenografts reached anapproximate volume of 1.5 cm³ and the tumors excised for histologicalexamination. This research project was approved by the Animal EthicsCommittee of the Austin and Repatriation Medical Centre.

Histological Examination of Tumor Xenografts

Xenografts were excised and bisected. One half was fixed in 10%formalin/PBS before being embedded in paraffin. Four micron sectionswere then cut and stained with haematoxylin and eosin (H&E) for routinehistological examination. The other half was embedded in Tissue Tek® OCTcompound (Sakura Finetek, Torrance, Calif.), frozen in liquid nitrogenand stored at −80° C. Thin (5 micron) cryostat sections were cut andfixed in ice-cold acetone for 10 min followed by air drying for afurther 10 min. Sections were blocked in protein blocking reagent(Lipshaw Immunon, Pittsburgh U.S.A.) for 10 min and then incubated withbiotinylated primary antibody (1 mg/ml), for 30 min at room temperature(RT). All antibodies were biotinylated using the ECL proteinbiotinylation module (Amersham, Baulkham Hills, Australia), as per themanufacturer's instructions. After rinsing with PBS, sections wereincubated with a streptavidin horseradish peroxidase complex for afurther 30 min (Silenus, Melbourne, Australia). Following a final PBSwash the sections were exposed to 3-amino-9-ethylcarbozole (AEC)substrate (0.1 M acetic acid, 0.1 M sodium acetate, 0.02 M AEC (SigmaChemical Co., St Louis, Mo.)) in the presence of hydrogen peroxide for30 min. Sections were rinsed with water and counterstained withhematoxylin for 5 min and mounted.

Efficacy of mAb806 in Preventative Model

mAb806 was examined for efficacy against U87MG and U87MG.Δ2-7 tumors ina preventative xenograft model. Antibody or vehicle were administeredi.p. the day before tumor inoculation and was given 3 times per week for2 weeks. mAb806 had no effect on the growth of parental U87MGxenografts, which express the wtEGFR, at a dose of 1 mg per injection(FIG. 9A). In contrast, mAb806 significantly inhibited the growth ofU87MG.Δ2-7 xenografts in a dose dependent manner (FIG. 9B). At day 20,when control animals were sacrificed, the mean tumor volume was1637±178.98 mm³ for the control group, a statistically smaller 526±94.74mm³ for the 0.1 mg per injection group (p<0.0001) and 197±42.06 mm³ forthe 1 mg injection group (p<0.0001). Treatment groups were sacrificed atday 24 at which time the mean tumor volumes was 1287±243.03 mm³ for the0.1 mg treated group and 492±100.8 mm³ for the 1 mg group.

Efficacy of mAb806 in Established Xenograft Model

Given the efficacy of mAb806 in the preventative xenograft model, itsability to inhibit the growth of established tumor xenografts was thenexamined. Antibody treatment was as described in the preventative modelexcept that it commenced when tumors had reached a mean tumor volume of65±6.42 mm³ for the U87MG.Δ2-7 xenografts and 84±9.07 mm³ for theparental U87MG xenografts. Once again, mAb806 had no effect on thegrowth of parental U87MG xenografts at a dose of 1 mg per injection(FIG.10A). In contrast, mAb806 significantly inhibited the growth ofU87MG.Δ2-7 xenografts in a dose dependent manner (FIG. 10B). At day 17,one day before control animals were sacrificed, the mean tumor volumewas 935±215.04 mm³ for the control group, 386±57.51 mm³ for the 0.1 mgper injection group (p<0.01) and 217±58.17 mm³ for the 1 mg injectiongroup (p<0.002).

To examine whether the growth inhibition observed with mAb806 wasrestricted to cell expressing de2-7 EGFR, its efficacy againstU87MG.wtEGFR tumor xenografts was examined in an established model.These cells serve as a model for tumors containing amplification of theEGFR gene without de2-7 EGFR expression. mAb806 treatment commenced whentumors had reached a mean tumor volume of 73±7.5 mm³. mAb806significantly inhibited the growth of established U87MG.wtEGFRxenografts when compared to control tumors treated with vehicle (FIG.10C). On the day control animals were sacrificed, the mean tumor volumewas 960±268.9 mm³ for the control group and 468±78.38 mm³ for the grouptreated with 1 mg injections (p<0.04).

Histological and Immunohistochemical Analysis of Established Tumors

To evaluate potential histological differences between mAb806-treatedand control U87MG.Δ2-7 and U87MG.wtEGFR xenografts (collected at days 24and 42 respectively), formalin-fixed, paraffin embedded sections werestained with H&E. Areas of necrosis were seen in sections from bothU87MG.Δ2-7 (collected 3 days after treatment finished), and U87MG.wtEGFRxenografts (collected 9 days after treatment finished) treated withmAb806. This result was consistently observed in a number of tumorxenografts (n=4). However, analysis of sections from xenografts treatedwith control did not display the same areas of necrosis seen with mAb806treatment. Sections from mAb806 or control treated U87MG xenografts werealso stained with H&E and revealed no differences in cell viabilitybetween the two groups, further supporting the hypothesis that mAb806binding induces decreased cell viability/necrosis within tumorxenografts.

An immunohistochemical analysis of U87MG, U87MG.Δ2-7 and U87MG.wtEGFRxenograft sections was performed to determine the levels of de2-7 andwtEGFR expression following mAb806 treatment. Sections were collected atdays 24 and 42 as above, and were immunostained with the 528 or 806antibodies. As expected, the 528 antibody stained all xenograft sectionswith no obvious decrease in intensity between treated and controltumors. Staining of U87MG sections was undetectable with the mAb806,however positive staining of U87MG.Δ2-7 and U87MG.wtEGFR xenograftsections was observed. There was no difference in mAb806 stainingdensity between control and treated U87MG.Δ2-7 and U87MG.wtEGFRxenografts suggesting that antibody treatment does not down regulatede2-7 or wtEGFR expression.

Treatment of A431 Xenografts with mAb806

To demonstrate that the anti-tumor effects of mAb806 were not restrictedto U87MG cells, the antibody was administered to mice with A431xenografts. These cells contain an amplified EGFR gene and expressapproximately 2×10⁶ receptors per cell. As described above, mAb806 bindsabout 10% of these EGFR and targets A431 xenografts. mAb806significantly inhibited the growth of A431 xenografts when examined inthe previously described preventative xenograft model (FIG. 11A). At day13, when control animals were sacrificed, the mean tumor volume was1385±147.54 mm³ in the control group and 260±60.33 mm³ for the 1 mginjection treatment group (p<0.0001).

In a separate experiment, a dose of 0.1 mg mAb also significantlyinhibited the growth of A431 xenografts in a preventative model.

Given the efficacy of mAb806 in the preventative A431 xenograft model,its ability to inhibit the growth of established tumor xenografts wasexamined. Antibody treatment was as described in the preventative modelexcept it was not started until tumors had reached a mean tumor volumeof 201±19.09 mm³. mAb806 significantly inhibited the growth ofestablished tumor xenografts (FIG. 11B). At day 13, when control animalswere sacrificed, the mean tumor volume was 1142±120.06 mm³ for thecontrol group and 451±65.58 mm³ for the 1 mg injection group (p<0.0001).

In summary, the therapy studies with mAb806 described here clearlydemonstrated dose dependent inhibition of U87MG.Δ2-7 xenograft growth.In contrast, no inhibition of parental U87MG xenografts was observeddespite the fact they continue to express the wtEGFR in vivo. mAb806 notonly significantly reduced xenograft volume, it also induced significantnecrosis within the tumor. This is the first report showing thesuccessful therapeutic use of such an antibody in vivo against a humande2-7 EGFR expressing glioma xenografts.

Gene amplification of the EGFR has been reported in a number ofdifferent tumors and is observed in approximately 50% of gliomas(Voldberg et al., 1997). It has been proposed that the subsequent EGFRover-expression mediated by receptor gene amplification may confer agrowth advantage by increasing intracellular signaling and cell growth(Filmus et al., 1987). The U87MG cell line was transfected with thewtEGFR in order to produce a glioma cell that mimics the process of EGFRgene amplification. Treatment of established U87MG.wtEGFR xenograftswith mAb806 resulted in significant growth inhibition. Thus, mAb806 alsomediates in vivo antitumor activity against cells containingamplification of the EGFR gene. Interestingly, mAb806 inhibition ofU87MG.wtEGFR xenografts appears to be less effective than that observedwith U87MG.Δ2-7 tumors. This probably reflects the fact that mAb806 hasa lower affinity for the amplified EGFR and only binds a smallproportion of receptors expressed on the cell surface. However, itshould be noted that despite the small effect on U87MG.wtEGFR xenograftvolumes, mAb806 treatment produced large areas of necrosis within thesexenografts.

To rule out the possibility that mAb806 only mediates inhibition of theU87MG derived cell lines we tested its efficacy against A431 xenografts.This squamous cell carcinoma derived cell line contains significant EGFRgene amplification which is retained both in vitro and in vivo.Treatment of A431 xenografts with mAb806 produced significant growthinhibition in both a preventative and established model, indicating theanti-tumor effects of mAb806 are not restricted to transfected U87MGcell lines.

EXAMPLE 11 Combination Therapy Treatment of A431 Xenografts with mAb806and AG1478

The anti-tumor effects of mAb806 combined with AG1478 was tested in micewith A431 xenografts. AG1478(4-(3-Chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selectiveinhibitor of the EGFR kinase versus HER2-neu and platelet-derived growthfactor receptor kinase (Calbiochem Cat. No. 658552). Three controls wereincluded: treatment with vehicle only, vehicle+mAb806 only, andvehicle+AG1478 only. The results are illustrated in FIG. 12. 0.1 mgmAb806 was administered at 1 day prior to xenograft and 1, 3, 6, 8 and10 days post xenograft. 400 μg AG1478 was administered at 0, 2, 4, 7, 9,and 11 days post xenograft.

Both AG1478 and mAb806, when administered alone, produced a significantreduction of tumor volume. However, in combination, the reduction oftumor volume was greatly enhanced.

In addition, the binding of mAb806 to EGFR of A431 cells was evaluatedin the absence and presence of AG1478. Cells were placed in serum freemedia overnight, then treated with AG1478 for 10 min at 37° C., washedtwice in PBS, then lysed in 1% Triton and lysates prepared bycentrifugation for 10 min at 12,000 g. Lysate was then assessed for 806reactivity by an ELISA in a modified version of an assay described bySchooler and Wiley, Analytical Biochemistry 277, 135-142 (2000). Plateswere coated with 10 μg/ml of mAb806 in PBS/EDTA overnight at roomtemperature and then washed twice. Plates were then blocked with 10%serum albumin/PBS for 2 hours at 37° C. and washed twice. A 1:20 celllysate was added in 10% serum albumin/PBS for 1 hour at 37° C., thenwashed four times. Anti-EGFR (SC-03; Santa Cruz Biotechnology Inc.) in10% serum albumin/PBS was reacted 90 min at room temperature, the platewashed four times, and anti-rabbit-HRP (1:2000 if from Silenus) in 10%serum albumin/PBS was added for 90 min at room temperature, washed fourtimes, and color developed using ABTS as a substrate. It was found thatmAb806 binding is significantly increased in the presence of increasingamounts of AG1478 (FIG. 13).

EXAMPLE 12 Immunoreactivity in Human Glioblastomas Pre-Typed for EGFRStatus

Given the high incidence of EGFR expression, amplification and mutationin glioblastomas, a detailed immunohistochemical study was performed inorder to assess the specificity of 806 in tumors other than xenografts.A panel of 16 glioblastomas was analyzed by immunohistochemistry. Thispanel of 16 glioblastomas was pre-typed by RT-PCR for the presence ofamplified wild-type EGFR and de2-7 EGFR expression. Six of these tumorsexpressed only the wtEGFR transcript, 10 had wtEGFR gene amplificationwith 5 of these showing wild-type EGFR transcripts only, and 5 bothwild-type EGFR and de2-7 gene transcript.

Immunohistochemical analysis was performed using 5mm sections of freshfrozen tissue applied to histology slides and fixed for 10 minutes incold acetone. Bound primary antibody was detected with biotinylatedhorse anti-mouse antibody followed by an avidin-biotin-complex reaction.Diaminobenzidine tetra hydrochloride (DAB) was used as chromogen. Theextent of the immunohistochemical reactivity in tissues was estimated bylight microscopy and graded according to the number of immunoreactivecells in 25% increments as follows:

Focal=less than 5%

+=5-25%

++=25-50%

+++=50-75%

++++=>75%

The 528 antibody showed intense reactivity in all tumors, while DH8.3immunostaining was restricted to those tumors expressing the de2-7 EGFR(Table 2). Consistent with the previous observations in FACS androsetting assays, mAb806 did not react with the glioblastomas expressingthe wtEGFR transcript from nonamplified EGFR genes (Table 2). Thispattern of reactivity for mAb806 is similar to that observed in thexenograft studies and again suggests that this antibody recognizes thede2-7 and amplified EGFR but not the wtEGFR when expressed on the cellsurface.

TABLE 2 Immunoreactivity of mAbs528, DH8.3 and 806 on glioblastomaspre-typed for the presence of wild-type EGFR and mutated de2-7 EGFR andfor their amplification status de2-7 EGFR Amplification Expression 528DH8.3 806 No ++++ — — No ++++ —  —* No ++++ — — No ++ — — No +++ — — No++++ — — Yes No ++++ — ++++ Yes No ++++ — + Yes No ++++ — +++ Yes No++++ — ++++ Yes No ++++ — +-++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++++++ ++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++ ++++ *focal staining

EXAMPLE 13 EGFR Immunoreactivity in Normal Tissue

In order to determine if the de2-7 EGFR is expressed in normal tissue,an immunohistochemical study with mAb806 and DH8.3 was conducted in apanel of 25 tissues. There was no strong immunoreactivity with eithermAb806 or DH8.3 in any tissue tested, suggesting that the de2-7 EGFR isabsent in normal tissues (Table 3). There was some variable stainingpresent in tonsils with mAb806 that was restricted to the basal celllayer of the epidermis and mucosal squamous cells of the epithelium. Inplacenta, occasional immunostaining of the trophoblast epithelium wasobserved. Interestingly, two tissues that express high endogenous levelsof wtEGFR, the liver and skin, failed to show any significant mAb806reactivity. No reactivity was observed with the liver samples at all,and only weak and inconsistent focal reactivity was detectedoccasionally (in no more than 10% of all samples studied) in basalkeratinocytes in skin samples and in the squamous epithelium of thetonsil mucosa, further demonstrating that this antibody does not bindthe wtEGFR expressed on the surface of cells to any significant extent(Table 3). All tissues were positive for the wtEGFR as evidenced by theuniversal staining seen with the 528 antibody (Table 3).

TABLE 3 Reactivity of 582, DH8.3 and 806 on normal tissues Tissue 528DH8.3 806 Esophagus pos — — Stomach pos — — Duodenum pos — — Small pos —— intestine/duodenum Colon pos — — Liver pos — — Salivary glands pos — —(parotid) Kidney pos — — Urinary Bladder pos — — Prostate pos — — Testispos — — Uterus (cx/endom) pos —* — Fallopian tube pos — — Ovary pos — —Breast pos —* — Placenta pos — — Peripheral nerve pos — — Skeletalmuscle pos — — Thyroid gland pos — — Lymph node pos — — Spleen pos — —Tonsil pos — — occ. weak reactivity of basal layer of squamousepithelium Heart pos — — Lung pos — — Skin pos — — occ. weak reactivityof basal layer of squamous epithelium *some stromal staining in varioustissue

EXAMPLE 14 EGFR Immunoreactivity in Various Tumors

The extent of de2-7 EGFR in other tumor types was examined using a panelof 12 different malignancies. The 528 antibody showed often homogeneousstaining in many tumors analyzed except melanoma and seminoma. Whenpresent, DH8.3 immunoreactivity was restricted to the occasional focaltumor cell indicating there is little if any de2-7 EGFR expression intumors outside the brain using this detection system (Table 4). Therewas also focal staining of blood vessels and a varying diffuse stainingof connective tissue with the DH8.3 antibody in some tumors (Table 4).This staining was strongly dependent on antibody concentration used andwas considered nonspecific background reactivity. The mAb806 showedpositive staining in 64% of head and neck tumors and 50% of lungcarcinomas (Table 4). There was little mAb806 reactivity elsewhereexcept in urinary tumors that were positive in 30% of cases.

Since the head and neck and lung cancers were negative for the DH8.3antibody the reactivity seen with the mAb in these tumors maybeassociated with EGFR gene amplification.

TABLE 4 Monoclonal antibodies 528, DH8.3 and 806 on tumor panel Tumor528 DH8.3 806 Malignant melanoma  0/10 0/10  0/10 metastases Urinarybladder (tcc, 10/10 0/10*  3/10* sqcc, adeno) (7x++++, 2x++++, 1x+)(2x++++, 1x++) Mammary gland  6/10 1/10  1/10 (3x++++, 3x++) (1x+) (foc)Head + neck cancer 11/11 0/11* 7/11 (sqcc) (1x+++-10x++++) (3x++++,3x+++, 1x+) Lung (sqcc, adeno, 12/12 0/12* 6/12 neuroend)(10x++++-1x+++) (3x++++ 3x+++) Leiomyosarcoma 5/5 0/5  0/5  (4x++++,1x+) Liposarcoma 5/5 0/5  0/5* (2x + 3x +++) Synovial sarcoma  4/5* 0/5 0/5* (4x ++++) Mfh Malignant  4/5* 0/5*  0/5* fibrous histiocytomaColonic carcinoma 10/10 0/10* 0/10 (9x++++, 1x+) Seminoma  1/10* 1/10*0/10 Ovary (serous- 4/5 0/5*  0/5  papillary) (3x++++, 1x+) *focalstaining

EXAMPLE 15 Immunoreactivity in Human Glioblastomas Unselected for EGFRStatus

In order to confirm the unique specificity and to evaluate thereactivity of mAb806, it was compared to the 528 and DH8.3 antibodies ina panel of 46 glioblastomas not preselected for their EGFR status. The528 antibody was strongly and homogeneously positive in all samplesexcept two (Nos. 27 and 29) (44/46, 95.7%). These two cases were alsonegative for mAb806 and mAbDH8.3. The mAb806 was positive in 27/46(58.7%) cases, 22 of which displayed homogeneous immunoreactivity inmore than 50% of the tumor. The DH8.3 antibody was positive in 15/46(32.6%) glioblastomas, 9 of which showed homogeneous immunoreactivity.The immunochemical staining of these unselected tumors is tabulated inTable 5.

There was concordance between mAb806 and DH8.3 in every case except one(No. 35). A molecular analysis for the presence of EGFR amplificationwas done in 44 cases (Table 5). Of these, 30 cases co-typed with thepreviously established mAb806 immunoreactivity pattern: e.g., 16mAb806-negative cases revealed no EGFR amplification and 14EGFR-amplified cases were also mAb806 immunopositive. However, 13 cases,which showed 806 immunoreactivity, were negative for EGFR amplificationwhile one EGFR-amplified case was mAb806 negative. Further analysis ofthe mutation status of these amplification negative and 806 positivecases is described below and provides explanation for most of the 13cases which were negative for EGFR amplification and were recognized by806.

Subsequently, a molecular analysis of the deletion mutation by RT-PCRwas performed on 41/46 cases (Table 5). Of these, 34 cases co-typed withDH8.3 specific for the deletion mutation: 12 cases were positive in bothRT-PCR and immunohistochemistry and 22 cases were negative/negative.Three cases (#2, #34, and #40) were DH8.3 positive/RT-PCR negative forthe deletion mutation and three cases (#12, #18, and #39) were DH8.3negative/RT-PCR positive. As expected based on our previous specificityanalysis, mAb806 immunoreactivity was seen in all DH8.3 positive tissuesexcept in one case (#35).

Case #3 also revealed a mutation (designated A2 in Table 5), whichincluded the sequences of the de2-7 mutation but this did not appear tobe the classical de2-7 deletion with loss of the 801 bases (data notshown). This case was negative for DH8.3 reactivity but showedreactivity with 806, indicating that 806 may recognize an additional andpossibly unique EGFR mutation.

TABLE 5 Immunohistochemical Analysis of 46 Unselected Glioblastomas WithmAbs 528, 806, and DH8.3 EGFR # 528 806 DH8.3 Amp. * 5′ MUT 1 ++++ ++++++ A 5′ MUT 2 ++++ ++++ ++++ N WT 3 ++++ ++++ neg. N A2 (det.) 4 ++++++++ neg. N WT 5 ++++ ++++ ++++ N 5′ MUT 6 ++++ ++++ neg. A WT 7 ++++++++ ++++ N 5′ MUT 8 ++++ ++++ ++++ A 5′ MUT 9 ++++ ++++ neg. A WT 10++++ neg. neg. N WT 11 ++ ++ ++ A 5′ MUT 12 ++++ ++ neg. A 5′ MUT 13++++ ++++ neg. N WT 14 ++ neg. neg. Nd nd 15 ++ ++ neg. N WT 16 + neg.neg. N nd 17 ++++ neg. neg. N WT 18 ++++ ++++ neg. A 5′ MUT 19 ++++ ++++neg. N WT 20 ++++ neg. neg. N WT 21 ++++ ++++ neg. N WT 22 +++ neg. neg.N WT 23 ++++ ++++ ++ N 5′ MUT 24 ++++ ++++ neg. A WT 25 ++++ neg. neg. NWT 26 ++++ ++++ +++ A 5′ MUT 27 neg. neg. neg. N WT 28 +++ neg. neg. NWT 29 neg. neg. neg. N WT 30 ++++ ++++ neg. N WT 31 par det neg. neg. Nnd 32 ++ +++ ++ N 5′ MUT 33 +++ ++++ ++++ A 5′ MUT 34 ++++ +++ ++++ N WT35 ++++ neg. ++++ A 5′ MUT 36 +++ ++ +++ A 5′ MUT 37 ++++ + + A 5′ MUT38 ++++ neg. neg. N WT 39 ++ neg. neg. N 5′ MUT 40 ++++ ++++ + A WT 41++ neg. neg. N WT 42 ++++ ++++ neg. A WT 43 ++++ neg. neg. nd nd 44 ++++neg. neg. N WT 45 ++++ neg. neg. N WT 46 ++++ neg. neg. N nd * N = notamplified, A—amplified, + WT = wild-type, 5′-mut nd = not done

The 806 antibody reactivity co-typed with amplified or de2-7 mutant EGFRin 19/27 or over 70% of the cases. It is notable that 2 of these 8 caseswere also DH8.3 reactive.

EXAMPLE 16 Systemic Treatment and Analysis of Intracranial Glioma Tumors

To test the efficacy of the anti-ΔEGFR monoclonal antibody, mAb806, wetreated nude mice bearing intracranial ΔEGFR-overexpressing gliomaxenografts with intraperitoneal injections of mAb806, the isotypecontrol IgG or PBS.

Because primary explants of human glioblastomas rapidly lose expressionof amplified, rearranged receptors in culture, no existing glioblastomacell lines exhibit such expression. To force maintenance of expressionlevels comparable with those seen in human tumors, U87MG, LN-Z308, andA1207 (gift from Dr. S. Aaronson, Mount Sinai Medical Center, New York,N.Y.) cells were infected with ΔEGFR, kinase-deficient ΔEGFR (DK), orwild-type EGFR (wtEGFR) viruses, which also conferred resistance to G418as described previously (Nishikawa et al. (1994) A mutant epidermalgrowth factor receptor common in human glioma confers enhancedtumorigenicity. Proc. Natl. Acad. Sci. U.S.A., 91, 7727-7731).

Populations expressing similar levels of the various EGFR alleles (theseexpression levels correspond approximately to an amplification level of25 gene copies; human glioblastomas typically have amplification levelsfrom 10 to 50 gene copies of the truncated receptor) were selected byFACS as described previously (Nishikawa et al., 1994) and designated asU87MG.ΔEGFR, U87MG.DK, U87MG.wtEGFR, LN-Z308.ΔEGFR, LN-Z308.DK,LN-Z308.wtEGFR, A1207.ΔEGFR, A1207.DK, and A1207.wtEGFR, respectively.Each was maintained in medium containing G418 (U87MG cell lines, 400μg/ml; LN-Z308 and A1207 cell lines, 800 μg/ml).

U87MG.ΔEGFR cells (1×10⁵) or 5×10⁵ LN-Z308.ΔEGFR, A1207.ΔEGFR, U87MG,U87MG.DK, and U87MG.wtEGFR cells in 5 μl of PBS were implanted into theright corpus stratum of nude mice brains as described previously(Mishima et al. (2000) A peptide derived from the non-receptor bindingregion of urokinase plasminogen activator inhibits glioblastoma growthand angiogenesis in vivo in combination with cisplatin. Proc. Natl.Acad. Sci. U.S.A. 97, 8484-8489). Systemic therapy with mAb806, or theIgG2b isotype control, was accomplished by i.p. injection of 1 μg ofmAbs in a volume of 100 μl every other day from post-implantation day 0through 14. For direct therapy of intracerebral U87MG.ΔEGFR tumors, 10μg of mAb806, or the IgG2b isotype control, in a volume of 5 μl wereinjected at the tumor-injection site every other day starting at day 1for 5 days.

Animals treated with PBS or isotype control IgG had a median survival of13 days, whereas mice treated with mAb806 had a 61.5% increase in mediansurvival up to 21 days (P<0.001; FIG. 24A).

Treatment of mice 3 days post-implantation, after tumor establishment,also extended the median survival of the mAb806-treated animals by 46.1%(from 13 days to 19 days; P<0.01) compared with that of the controlgroups (data not shown).

To determine whether these antitumor effects of mAb806 extended beyondU87MG.ΔEGFR xenografts, similar treatments were administered to animalsbearing other glioma cell xenografts of LN-Z308.ΔEGFR and A1207.ΔEGFR.The median survival of mAb806-treated mice bearing LN-Z308.ΔEGFRxenografts was extended from 19 days for controls to 58 days (P<0.001;FIG. 24B). Remarkably, four of eight mAb806-treated animals survivedbeyond 60 days (FIG. 24B). The median survival of animals bearingA1207.ΔEGFR xenografts was also extended from 24 days for controls to 29days (P<0.01; data not shown).

mAb806 Treatment Inhibits ΔEGFR-Overexpressing Brain Tumor Growth

Mice bearing U87MG.ΔEGFR and LN-Z308.ΔEGFR xenografts were euthanized atday 9 and day 15, respectively. Tumor sections were histopathologicallyanalyzed and tumor volumes were determined. Consistent with the resultsobserved for animal survival, mAb806 treatment significantly reduced thevolumes by about 90% of U87MG.ΔEGFR. (P<0.001; FIG. 24C) andLN-Z308.ΔEGFR by more than 95% (P<0.001; FIG. 24D) xenografts incomparison to that of the control groups. Similar results were obtainedfor animals bearing A1207.ΔEGFR tumors (65% volume reduction, P<0.01;data not shown).

Intratumoral Treatment with mAb806 Extends Survival of Mice BearingU87MG.ΔEGFR Brain Tumors

The efficacy of direct intratumoral injection of mAb806 for thetreatment of U87MG.ΔEGFR xenografts was also determined. Animals weregiven intratumoral injections of mAb806 or isotype control IgG one daypost-implantation. Control animals survived for 15 days, whereas mAb806treated mice remained alive for 18 days (P<0.01; FIG. 24E). While theintratumoral treatment with mAb806 was somewhat effective, it entailedthe difficulties of multiple intracranial injections and increased riskof infection. We therefore focused on systemic treatments for furtherstudies.

mAb806 Treatment Slightly Extends Survival of Mice Bearing U87MG.wtEGFRbut not U87MG or U87MG.DK Intracranial Xenografts

To determine whether the growth inhibition by mAb806 was selective fortumors expressing ΔEGFR, we treated animals bearing U87MG, U87MG.DK(kinase deficient ΔEGFR) and U87MG.wtEGFR brain xenografts. mAb806treatment did not extend survival of mice implanted with U87MG tumors(FIG. 25A) which expressed a low level of endogenous wild-type EGFR(wtEGFR) (Huang et al. (1997) The enhanced tumorigenic activity of amutant epidermal growth factor receptor common in human cancers ismediated by threshold levels of constitutive tyrosine phosphorylationand unattenuated signaling. J. Biol. Chem., 272, 2927-2935), or animalsbearing U87MG.DK xenografts which overexpressed a kinase-deficient ΔEGFRin addition to a low level of endogenous wtEGFR (FIG. 25B). The mAb806treatment slightly extended the survival of mice bearing U87MG.wtEGFRtumors (P<0.05, median survival 23 days versus 26 days for the controlgroups) which overexpressed wtEGFR (FIG. 25C).

mAb806 Reactivity Correlates with In vivo Anti-Tumor Efficacy

To understand the differential effect of mAb806 on tumors expressingvarious levels or different types of EGFR, we determined mAb806reactivity with various tumor cells by FACS analysis. Stained cells wereanalyzed with a FACS Calibur using Cell Quest software (Becton-DickinsonPharMingen). For the first antibody, the following mAbs were used:mAb806, anti EGFR mAb clone 528, and clone EGFR. 1. Mouse IgG2a or IgG2bwas used as an isotype control.

Consistent with previous reports (Nishikawa et al. (1994) A mutantepidermal growth factor receptor common in human glioma confers enhancedtumorigenicity. Proc. Natl. Acad. Sci. U.S.A., 91, 7727-7731), theanti-EGFR mAb528 recognized both ΔEGFR and wtEGFR and demonstratedstronger staining for U87MG.ΔEGFR cells compared with U87MG cells (FIG.26A, 528).

In contrast, antibody EGFR.1 reacted with wtEGFR but not with ΔEGFR(Nishikawara et al., 1994), because U87MG.ΔEGFR cells were as weaklyreactive as U87MG cells (FIG. 26A, panel EGFR.1).

This EGFR.1 antibody reacted with U87MG.wtEGFR more intensively thanwith U87MG cells, because U87MG.wtEGFR cells overexpressed wtEGFR (FIG.26A, panel EGFR.1). Although mAb806 reacted intensely with U87MG.ΔEGFRand U87MG.DK cells and not with U87MG cells, it reacted weakly withU87MG.wtEGFR, which indicated that mAb806 is selective for ΔEGFR with aweak cross-activity to overexpressed wtEGFR (FIG. 26A, panel mAb806).

This level of reactivity with U87MG.wtEGFR was quantitatively andqualitatively similar to the extension of survival mediated by theantibody treatment (FIG. 25C).

We further determined mAb806 specificity by immunoprecipitation. EGFRsin various cell lines were immunoprecipitated with antibodies mAb806,anti-EGFR mAb clone 528 (Oncogene Research Products, Boston, Mass.), orclone EGFR.1 (Oncogene Research Products).

Briefly, cells were lysed with lysis buffer containing 50 mM HEPES (pH7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 0.1% SDS,0.5% sodium deoxycholate, 10 mM sodium PPi, 1 mM phenylmethlsulfonylfluoride, 2 mM Na3 V0₄, 5 μg/ml leupeptin, and 5 μg/ml aprotinin.Antibodies were incubated with cell lysates at 4° C. for 1 h before theaddition of protein-A and-G Sepharose. Immunoprecipitates were washedtwice with lysis buffer and once with HNTG buffer [50 mM HEPES (pH 7.5),150 mM NaCl, 0.1% Triton X-100, and 10% glycerol], electrophoresed, andtransferred to nitrocellulose membranes.

Blots of electrophoretically-separated proteins were probed with theanti-EGFR antibody, C13 (provided by Dr. G. N. Gill, University ofCalifornia, San Diego, Calif.), used for detection of both wild-type andΔEGFR on immunoblots (Huang et al., 1997), and proteins were visualizedusing the ECL chemiluminescent detection system (Amersham PharmaciaBiotech.). Antibodies to Bcl-X (rabbit poly-clonal antibody;Transduction Laboratories, Lexington, Ky.) and phosphotyrosine (4G10,Upstate Biotechnology, Lake Placid, N.Y.) were used for Western blotanalysis as described previously (Nagane et al. (1998) Drug resistanceof human glioblastoma cells conferred by a tumor-specific mutantepidermal growth factor receptor through modulation of Bcl-XL andcaspase-3-like proteases. Proc. Natl. Acad. Sci. U.S.A. 95, 5724-5729).

Consistent with the FACS analysis, antibody 528 recognized wtEGFR andmutant receptors (FIG. 26B-panel IP: 528), whereas antibody EGFR.1reacted with wtEGFR but not with the mutant species (FIG. 26B, panelIP:EGFR.1). Moreover, the levels of mutant receptors in U87MG.ΔEGFR andU87MG.DK cells are comparable with those of wtEGFR in the U87MG.wtEGFRcells (FIG. 26B, panel IP: 528).

However, antibody mAb806 was able to precipitate only a small amount ofthe wtEGFR from the U87MG.wtEGFR cell lysates as compared with thelarger amount of mutant receptor precipitated from U87MG.ΔEGFR andU87MG.DK cells, and an undetectable amount from the U87MG cells (FIG.26B, panel IP:mAb806). Collectively, these data suggest that mAb806recognizes an epitope in ΔEGFR that also exists in a small fraction ofwtEGFR only when it is overexpressed on the cell surface (see furtherdiscussion of and references to the mAb806 epitope below).

mAb806 Treatment Reduces ΔEGFR Autophosphorylation and Down-RegulatesBcl.X_(L) Expression in U87MG.ΔEGFR Brain Tumors

The mechanisms underlying the growth inhibition by mAb806 were nextinvestigated. Since the constitutively active kinase activity andautophosphorylation of the carboxyl terminus of ΔEGFR are essential forits biological functions (Nishikawa et al. (1994) A mutant epidermalgrowth factor receptor common in human glioma confers enhancedtumorigenicity. Proc. Natl. Acad. Sci. U.S.A. 91, 7727-7731; Huang etal., 1997; Nagane et al. (1996) A common mutant epidermal growth factorreceptor confers enhanced tumorigenicity on human glioblastoma cells byincreasing proliferation and reducing apoptosis. Cancer Res., 56,5079-5086; Nagane et al. (2001) Aberrant receptor signaling in humanmalignant gliomas: mechanisms and therapeutic implications. Cancer Lett.162 (Suppl.1), S17-S21) ΔEGFR phosphorylation status was determined intumors from treated and control animals. As shown in FIG. 27A, mAb806treatment dramatically reduced ΔEGFR autophosphorylation, althoughreceptor levels were only slightly decreased in the mAb806-treatedxenografts. We have previously shown that receptor autophosphorylationcauses up-regulation of the antiapoptotic gene, Bcl-X_(L), which plays akey role in reducing apoptosis of ΔEGFR-overexpressing tumors (Nagane etal., 1996; Nagane et al., 2001). Therefore, the effect of mAb806treatment on Bcl-X_(L) expression was next determined. ΔEGFR tumors frommAb806-treated animals did indeed show reduced levels of Bcl-X_(L) (FIG.27A).

mAb806 Treatment Decreases Growth and Angiogenesis, and IncreasesApoptosis in U87MG.ΔEGFR Tumors

In light of the in vivo suppression caused by mAb806 treatment and itsbiochemical effects on receptor signaling, we determined theproliferation rate of tumors from control or treated mice. Theproliferative index, measured by Ki-67 staining of the mAb806-treatedtumors, was significantly lower than that of the control tumors(P<0.001; FIG. 28).

Briefly, to assess angiogenesis in tumors, they were fixed in a solutioncontaining zinc chloride, paraffin embedded, sectioned, andimmunostained using a monoclonal rat anti-mouse CD31 antibody(Becton-Dickinson PharMingen; 1:200). Assessment of tumor cellproliferation was performed by Ki-67 immunohistochemistry onformalin-fixed paraffin-embedded tumor tissues. After deparaffinizationand rehydration, the tissue sections were incubated with 3% hydrogenperoxide in methanol to quench endogenous peroxidase. The sections wereblocked for 30 min with goat serum and incubated overnight with theprimary antibody at 4° C. The sections were then washed with PBS andincubated with a biotinylated secondary antibody for 30 min. Afterseveral washes with PBS, products were visualized using streptavidinhorseradish peroxidase with diaminobenzidine as chromogen andhematoxylin as the counterstain. As a measure of proliferation, theKi-67 labeling index was determined as the ratio of labeled: totalnuclei in high-power (3400) fields.

Approximately 2000 nuclei were counted in each case by systematic randomsampling. For macrophage and NK cell staining, frozen sections, fixedwith buffered 4% paraformaldehyde solution, were immunostained usingbiotinylated mAbF4/80 (Serotec, Raleigh, N.C.) and polyclonal rabbitanti-asialo GM1 antibody (Dako Chemicals, Richmond, Va.), respectively.Angiogenesis was quantitated as vessel area using computerized analysis.For this purpose, sections were immunostained using anti-CD31 and wereanalyzed using a computerized image analysis system withoutcounterstain. MVAs were determined by capturing digital images of thesections at 3200 magnification using a CCD color camera as describedpreviously (Mishima et al., 2000). Images were then analyzed using ImagePro Plus version 4.0 software (Media Cybernetics, Silver Spring, Md.)and MVA was determined by measuring the total amount of staining in eachsection. Four fields were evaluated for each slide. This value wasrepresented as a percentage of the total area in each field. Resultswere confirmed in each experiment by at least two observers (K. M., H-J.S. H.).

In addition, apoptotic cells in tumor tissue were detected by using theTUNEL method as described previously (Mishima et al., 2000).TUNEL-positive cells were counted at X400. The apoptotic index wascalculated as a ratio of apoptotic cell number: total cell number ineach field. Analysis of the apoptotic index through TUNEL stainingdemonstrated a significant increase in the number of apoptotic cells inmAb806 treated tumors as compared with the control tumors (P<0.001; FIG.28).

The extent of tumor vascularization was also analyzed by immunostainingof tumors from treated and control specimens for CD31. To quantify tumorvascularization, microvascular areas (MVAs) were measured usingcomputerized image analysis. mAb806-treated tumors showed 30% less MVAthan did control tumors (P<0.001; FIG. 28).

To understand whether interaction between receptor and antibody mayelicit an inflammatory response, we stained tumor sections for themacrophage marker, F4/80, and the NK cell marker, asialo GM1.Macrophages were identified throughout the tumor matrix and especiallyaccumulated around the mAb806-treated-U87MG.ΔEGFR-tumor periphery (FIG.28). We observed few NK cells infiltrated in and around the tumors andno significant difference between mAb806-treated and isotype-controltumors (data not shown).

EXAMPLE 17 Combination Immunotherapy with mAb806 and mAb528

The experiments set forth herein describe in vivo work designed todetermine the efficacy of antibodies in accordance with this invention.

Female nude mice, 4-6 weeks old, were used as the experimental animals.Mice received subscutaneous inoculations of 3×10⁶ tumor cells in each oftheir flanks

The animals received either U87MG.D2-7, U87MG.DK, or A431 cells, all ofwhich are described, supra. Therapy began when tumors had grown to asufficient size.

Mice then received injections of one of (i) phosphate buffered saline,(ii) mAb806 (0.5 mg/injection), (iii) mAb528 (0.5mg/injection), or (iv)a combination of both mAbs. With respect to “(iv),” different groups ofmice received either 0.5 mg/injection of each mAb, or 0.25 mg/injectionof each mAb.

The first group of mice examined were those which had receivedU87MG.D2-7 injections. The treatment protocol began 9 days afterinoculation, and continued 3 times per week for 2 weeks (i.e., theanimals were inoculated 9, 11, 13, 16, 18 and 20 days after they wereinjected with the cells). At the start of the treatment protocol, theaverage tumor diameter was 115 mm³. Each group contained 50 mice, eachwith two tumors.

Within the group of mice which received the combination of antibodies(0.5 mg/injection of each), there were three complete regressions. Therewere no regressions in any of the other groups. FIG. 18A shows theresults graphically.

In a second group of mice, the injected materials were the same, exceptthe combination therapy contained 0.25 mg of each antibody perinjection. The injections were given 10, 12, 14, 17, 19 and 21 daysafter inoculation with the cells. At the start of the therapy theaverage tumor size was 114 mm³. Results are shown in FIG. 18B.

The third group of mice received inoculations of U87MG.DK. Therapeuticinjections started 18 days after inoculation with the cells, andcontinued on days 20, 22, 25, 27 and 29. The average tumor size at thestart of the treatment was 107 mm³. FIG. 18C summarizes the results. Thetherapeutic injections were the same as in the first group.

Finally, the fourth group of mice, which had been inoculated with A431cells, received injections as in groups I and III, at 8, 10, 12 and 14days after inoculation. At the start, the average tumor size was 71 mm³.Results are shown in FIG. 18D.

The results indicated that the combination antibody therapy showed asynergistic effect in reducing tumors. See FIG. 18A. A similar effectwas seen at a lower dose, as per FIG. 18B, indicating that the effect isnot simply due to dosing levels.

The combination therapy did not inhibit the growth of U87MG.DK (FIG.18C), indicating that antibody immune function was not the cause for thedecrease seen in FIGS. 18A and 18B.

It is noted that, as shown in FIG. 18D, the combination therapy alsoexhibited synergistic efficacy on A431 tumors, with 4 doses leading to a60% complete response rate. These data suggest that the EGFR moleculerecognized by mAb806 is functionally different from that inhibited by528.

EXAMPLE 18 mAb806 Inhibition of Tumor Xenografts Growth

As discussed herein, and further demonstrated and discussed in thisExample, mAb806 has been unexpectedly been found to inhibit the growthof tumor xenographs expressing either de2-7 or amplified EGFR, but notwild-type EGFR

Cell lines and antibodies were prepared as described in Example 1. Todetermine the specificity of mAb806, its binding to U87MG, U87MG.D2-7,and U87MG.wtEGFR cells was analyzed by FACS. Briefly, cultured parentaland transfected U87MG cell lines were analyzed for wild-type andde2-7EGFR expression using the 528, 806, and DH8.3 antibodies. Cells (13 10 6) were incubated with 5 μg/ml of the appropriate antibody or anisotype-matched negative control in PBS containing 1% HSA for 30 min at4° C. After three washes with PBS/1% HSA, cells were incubated anadditional 30 min at 4° C. with FTTC-coupled goat anti-mouse antibody(1:100 dilution; Calbiochem, San Diego, Calif.). After three subsequentwashes, cells were analyzed on an Epics Elite ESP (Beckman Coulter,Hialeah, Fla.) by observing a minimum of 20,000 events and analyzedusing EXPO (version 2) for Windows. An irrelevant IgG2b (mAb 100-310directed to the human antigen A33) was included as an isotype controlfor mAb806, and the 528 antibody was included because it recognizes boththe de2-7 and wtEGFR.

Only the 528 antibody was able to stain the parental U87MG cell line(FIG. 29), consistent with previous reports demonstrating that thesecells express the wtEGFR (Nishikawa et al. (1994) A mutant epidermalgrowth factor receptor common in human glioma confers enhancedtumorigenicity. Proc. Natl. Acad. Sci. U.S.A. 91, 7727-7731). mAb806 hadbinding levels similar to the control antibody, clearly demonstratingthat it is unable to bind the wtEGFR (FIG. 29). Binding of the isotypecontrol antibody to the U87MG.D2-7 and U87MG.wtEGFR cell lines wassimilar to that observed for the U87MG cells. mAb806 stained U87MG.D2-7and U87MG.wtEGFR cells, indicating that mAb806 specifically recognizedthe de2-7 EGFR and a subset of the overexpressed EGFR (FIG. 29). Asexpected, the 528 antibody stained both the U87MG.D2-7 and U87MG.wtEGFRcell lines (FIG. 29). The intensity of 528 antibody staining onU87MG.wtEGFR cells was much higher than mAb806, suggesting that mAb806only recognizes a portion of the overexpressed EGFR. The mAb806reactivity observed with U87MG.wtEGFR cells is similar to that obtainedwith A431 cells, another cell line that over expresses the wtEGFR.3

A Scatchard analysis was performed using U87MG.D2-7 and A431 cells todetermine the relative affinity and binding sites for mAb806 on eachcell line. mAb806 had an affinity for the de2-7EGFR receptor of1.1×10⁹M⁻¹ and recognized an average (three separate experiments) of2.4×10⁵ binding sites/cell, as noted in Example 4. In contrast, theaffinity of mAb806 for the wtEGFR on A431 cells was only 9.5×10⁷M⁻¹, asnoted in Example 8. Interestingly, mAb806 recognized 2.3×10⁵ bindingsites on the surface of A431, which is some 10-fold lower than thereported number of EGFR found in these cells. To confirm the number ofEGFR on the surface of our A431 cells, we performed a Scatchard analysisusing ¹²⁵I-labeled 528 antibody. As expected, this antibody bound toapproximately 2×10⁶ sites on the surface of A431 cells. Thus, it appearsthat mAb806 only binds a portion of the EGFR receptors on the surface ofA431 cells. Importantly, ¹²⁵I-labeled mAb806 did not bind to theparental U87MG cells at all, even when the number of cells was increasedto 1×10⁷.

mAb806 reactivity was further characterized in the various cell lines byimmunoprecipitation after ³⁵S-labeling using mAb806, sc-03 (a commercialpolyclonal antibody specific for the COOH-terminal domain of the EGFR)and a IgG2b isotype control. Briefly, cells were labeled for 16 h with100 mCi/ml of Tran ³⁵S-Label (ICN Biomedicals, Irvine, Calif.) in DMEMwithout methionine/cysteine supplemented with 5% dialyzed FCS. Afterwashing with PBS, cells were placed in lysis buffer (1% Triton X-100, 30mM HEPES, 150 mM NaCl, 500 μM 4-(2-aminoethyl)benzenesulfonylfluoride(AEBSF), 150 nM aprotinin, 1 μM E-64 protease inhibitor, 0.5 mM EDTA,and 1 μM leupeptin, pH 7.4) for 1 h at 4° C. Lysates were clarified bycentrifugation for 10 min at 12,000 g and then incubated with 5 μg ofappropriate antibody for 30 min at 4° C. before the addition of ProteinA-Sepharose. Immunoprecipitates were washed three times with lysisbuffer, mixed with SDS sample buffer, separated by gel electrophoresisusing a 4-20% Tris/glycine gel that was then dried, and exposed to X-rayfilm.

The sc-03 antibody immunoprecipitated three bands from U87MG.Δ2-7 cells;a doublet corresponding to the 2 de2-7 EGFR bands observed in thesecells and a higher molecular weight band corresponding to the wtEGFR(FIGS. 22 and 30). In contrast, while mAb806 immunoprecipitated the twode2-7 EGFR bands, the wtEGFR was completely absent. The pattern seen inU87MG.wtEGFR and A431 cells was essentially identical. The sc-03antibody immunoprecipitated a single band corresponding to the wtEGFRfrom A431 cells (FIGS. 22 and 30). mAb806 also immunoprecipitated asingle band corresponding to the wtEGFR from both U87MG.wtEGFR and A431cells (FIGS. 22 and 30). Consistent with the FACS and Scatchard data,the amount of EGFR immunoprecipitated by mAb806 was substantially lessthan the total EGFR present on the cell surface. Given that mAb806 andthe sc-03 immunoprecipitated similar amounts of the de2-7 EGFR, thisresult supports the notion that the mAb806 antibody only recognizes aportion of the EGFR in cells overexpressing the receptor. Comparisonsbetween mAb806 and the 528 antibody showed an identical pattern ofreactivity (data not shown). An irrelevant IgG2b (an isotype control formAb806) did not immunoprecipitate EGFR from either cell line (FIGS. 22and 30). Using identical conditions, mAb806 did not immunoprecipitatethe EGFR from the parental U87MG cells (data not shown).

mAb806 was also examined for efficacy against U87MG and U87MG.Δ2-7tumors in a preventative xenograft model. Antibody or vehicle wasadministered i.p. the day before tumor inoculation and was given threetimes per week for 2 weeks. At a dose of 1 mg/injection, mAb806 had noeffect on the growth of parental U87MG xenografts that express thewtEGFR (FIG. 9A). In contrast, mAb806 inhibited significantly the growthof U87MG.Δ2-7 xenografts in a dose-dependent manner (FIG. 9B). Twentydays after tumor inoculation, when control animals were sacrificed, themean tumor volume was 1600±180 mm³ for the control group, asignificantly smaller 500±95 mm³ for the 0.1 mg/injection group(P<0.0001) and 200±42 mm³ for the 1 mg/injection group (P<0.0001).Treatment groups were sacrificed at day 24, at which time the mean tumorvolumes were 1300±240 mm³ for the 0.1 mg treated group and 500±100 mm³for the 1 mg group (P<0.005).

Given the efficacy of mAb806 in the preventative xenograft model, itsability to inhibit the growth of established tumor xenografts wasexamined. Antibody treatment was as described in the preventative model,except that it commenced when tumors had reached a mean tumor volume of65 mm³ (10 days after implantation) for the U87MG.Δ2-7 xenografts and 84mm³ (19 days after implantation) for the parental U87MG xenografts (seeExample 10). Once again, mAb806 had no effect on the growth of parentalU87MG xenografts, even at a dose of 1 mg/injection (FIG. 10A). Incontrast, mAb806 significantly inhibited the growth of U87MG.Δ2-7xenografts in a dose-dependent manner (FIG. 10B). At day 17, one daybefore control animals were sacrificed, the mean tumor volume was900±200 mm³ for the control group, 400±60 mm³ for the 0.1 mg/injectiongroup (P<0.01), and 220±60 mm³ for the 1 mg/injection group (P<0.002).Treatment of U87MG.Δ2-7 xenografts with an IgG2b isotype control had noeffect on tumor growth (data not shown).

To examine whether the growth inhibition observed with mAb806 wasrestricted to cells expressing de2-7 EGFR, its efficacy against theU87MG.wtEGFR xenografts was also examined in an established model. Thesecells serve as a model for tumors containing amplification of the EGFRgene without de2-7 EGFR expression. mAb806 treatment commenced whentumors had reached a mean tumor volume of 73 mm³ (22 days afterimplantation). mAb806 significantly inhibited the growth of establishedU87MG.wtEGFR xenografts when compared with control tumors treated withvehicle (FIG. 10C). On the day control animals were sacrificed, the meantumor volume was 1000±300 mm³ for the control group and 500±80 mm³ forthe group treated with 1 mg/injection (P<0.04).

To evaluate potential histological differences between mAb806-treatedand control U87MG.Δ2-7 and U87MG.wtEGFR xenografts, formalin-fixed,paraffin-embedded sections were stained with H&E (FIG. 31). Areas ofnecrosis were seen in sections from mAb806-treated U87MG.Δ2-7(mAb806-treated xenografts were collected 24 days after tumorinoculation and vehicle treated xenografts at 18 days), and U87MG.wtEGFRxenografts (mAb806 xenografts were collected 42 days after tumorinoculation and vehicle treated xenografts at 37 days; FIG. 31). Thisresult was consistently observed in a number of tumor xenografts (n=4for each cell line). However, sections from U87MG.Δ2-7 and U87MG.wtEGFRxenografts treated with vehicle (n=5) did not display the same areas ofnecrosis seen after mAb806 treatment (FIG. 31). Vehicle andmAb806-treated xenografts removed at identical times also showed thesedifferences in tumor necrosis (data not shown). Thus, the increase innecrosis observed was not caused by the longer growth periods used forthe mAb806-treated xenografts. Furthermore, sections from mAb806-treatedU87MG xenografts were also stained with H&E and did not reveal any areasof necrosis (data not shown), further supporting the hypothesis thatmAb806 binding induces decreased cell viability, resulting in increasednecrosis within tumor xenografts.

An immunohistochemical analysis of U87MG, U87MG.Δ2-7, and U87MG.wtEGFRxenograft sections was performed to determine the levels of de2-7 andwtEGFR expression after mAb806 treatment (FIG. 32). As expected, the 528antibody stained all xenografts sections with no obvious decrease inintensity between treated and control tumors (FIG. 32). Staining ofU87MG sections was undetectable with the mAb806; however, positivestaining of U87MG.Δ2-7 and U87MG.wtEGFR xenograft sections was observed(FIG. 32). There was no difference in mAb806 staining intensity betweencontrol and treated U87MG.Δ2-7 and U87MG.wtEGFR xenografts, suggestingthat antibody treatment does not lead to the selection of clonalvariants lacking mAb806 reactivity.

To demonstrate that the antitumor effects of mAb806 were not restrictedto U87MG cells, the antibody was administrated to mice containing A431xenografts. These cells contain an amplified EGFR gene and expressapproximately 2×10⁶ receptors/cells. We have previously shown thatmAb806 binds ˜10% of these EGFRs and targets A431 xenografts (Garcia etal. (1993) Expression of mutated epidermal growth factor receptor bynon-small cell along carcinomas. Cancer Res. 53, 3217-3220). mAb806significantly inhibited the growth of A431 xenografts when examined inthe preventative xenograft model described previously (FIG. 11A). At day13, when control animals were sacrificed, the mean tumor volume was1400±150 mm³ in the vehicle-treated group and 260±60 mm³ for the 1mg/injection treatment group (P<0.0001). In a separate experiment, adose of 0.1 mg of mAb also inhibited significantly (P<0.05) the growthof A431 xenografts in a preventative model (data not shown) (see Example10).

Given the efficacy of mAb806 in the preventative A431 xenograft model,its ability to inhibit the growth of established tumor xenografts wasexamined. Antibody treatment was as described in the preventative model,except it was not started until tumors had reached a mean tumor volumeof 200±20 mm³. mAb806 significantly inhibited the growth of establishedA431 xenografts (FIG. 11B). At day 13, the day control animals weresacrificed, the mean tumor volume was 1100±100 mm³ for the control groupand 450±70 mm³ for the 1 mg/injection group (P<0.0001).

EXAMPLE 19 Construction, Expression and Analysis of Chimeric 806Antibody

Chimeric antibodies are a class of molecules in which heavy and lightchain variable regions of for instance, a mouse, rat or other speciesare joined onto human heavy and light chain regions, Chimeric antibodiesare produced recombinantly. One advantage of chimeric antibodies is thatthey can reduce xenoantigenic effects, the inherent immunogenicity ofnon-human antibodies (for instance, mouse, rat or other species). Inaddition, recombinantly prepared chimeric antibodies can often beproduced in large quantities, particularly when utilizing high levelexpression vectors.

For high level production, the most widely used mammalian expressionsystem is one which utilizes the gene amplification procedure offered bydehydrofolate reductase deficient (“dhfr-”) Chinese hamster ovary cells.The system is well known to the skilled artisan. The system is basedupon the dehydrofolate reductase “dhfr” gene, which encodes the DHFRenzyme, which catalyzes conversion of dehydrofolate to tetrahydrofolate.In order to achieve high production, dhfr-CHO cells are transfected withan expression vector containing a functional DHFR gene, together with agene that encodes a desired protein. In this case, the desired proteinis recombinant antibody heavy chain and/or light chain.

By increasing the amount of the competitive DHFR inhibitor methotrexate(MTX), the recombinant cells develop resistance by amplifying the dhfrgene. In standard cases, the amplification unit employed is much largerthan the size of the dhfr gene, and as a result the antibody heavy chainis co-amplified.

When large scale production of the protein, such as the antibody chain,is desired, both the expression level, and the stability of the cellsbeing employed, are critical. In long term culture, recombinant CHO cellpopulations lose homogeneity with respect to their specific antibodyproductivity during amplification, even though they derive from asingle, parental clone.

Bicistronic expression vectors were prepaid for use in recombinantexpression of the chimeric antibodies. These bicistronic expressionvectors, employ an “internal ribosomal entry site” or “IRES.” In theseconstructs for production of chimeric anti-EGFR, the immunoglobulinchains and selectable markers cDNAs are linked via an IRES. IRES arecis-acting elements that recruit the small ribosomal subunits to aninternal initiator codon in the mRNA with the help of cellulartrans-acting factors. IRES facilitate the expression of two or moreproteins from a polycistronic transcription unit in eukaryotic cells.The use of bicistronic expression vectors in which the selectable markergene is translated in a cap dependent manner, and the gene of interestin an IRES dependent manner, has been applied to a variety ofexperimental methods. IRES elements have been successfully incorporatedinto vectors for cellular transformation, production of transgenicanimals, recombinant protein production, gene therapy, gene trapping,and gene targeting.

Synopsis of Chimeric Antibody 806 (ch806) Construction

The chimeric 806 antibody was generated by cloning the VH and VL chainsof the 806 antibody from the parental murine hybridoma using standardmolecular biology techniques. The VH and VL chains were then cloned intothe pREN mammalian expression vectors, the construction of which are setforth in SEQ ID NO:7 and SEQ ID NO:8, and transfected into CHO(DHFR−/−ve) cells for amplification and expression. Briefly, followingtrypsinization 4×10⁶ CHO cells were co-transferred with 10 μg of each ofthe LC and HC expression vectors using electroporation under standardconditions. Following a 10 min rest period at room temperature, thecells were added to 15 ml medium (10% fetal calf serum,hypoxanthine/thymidine supplement with additives) and transferred to15×10 cm cell culture petri dishes. The plates were then placed into theincubator under normal conditions for 2 days.

At this point, the addition of gentamycin, 5 nM methotrexate, thereplacement of fetal calf serum with dialyzed fetal calf serum and theremoval of hypoxanthine/thymidine, initiated the selection for clonesthat were successfully transfected with both the LC and HC from themedium. At day 17 following transfection, individual clones growingunder selection were picked and screened for expression of the chimeric806 antibody. An ELISA was utilized for screening and consisted ofcoating an ELISA plate with denatured soluble EGF receptor (denaturedEGFR is known to allow 806 binding). This assay allows for the screeningof production levels by individual clones and also for the functionalityof the antibody being screened. All clones were shown to be producingfunctional ch806 and the best producer was taken and expanded foramplification. To amplify the level of ch806 being produced, the highestproducing clone was subjected to reselection under a higher methotrexateconcentration (100 nM vs. 5 nM). This was undertaken using theaforementioned procedures.

Clones growing at 100 nM MTX were then passed onto the BiologicalProduction Facility, Ludwig Institute, Melbourne, Australia formeasurement of production levels, weaning off serum, cell banking Thecell line has been shown to stably produce ˜10 mg/litre in rollerbottles.

The nucleic acid sequence of the pREN ch806 LC neo vector is provided inSEQ ID NO:7. The nucleic acid sequence of the pREN ch806 HC DHFR vectoris provided in SEQ ID NO:8.

FIG. 33 depicts the vectors pREN-HC and pREN-LC, which employ an IRES.The pREN bicistronic vector system is described and disclosed in U.S.Patent Application No. 60/355,838 filed Feb. 13, 2002, which isincorporated herein by reference in its entirety.

ch806 was assessed by FACS analysis to demonstrate that the chimeric 806displays identical binding specificity to that of the murine parentalantibody. Analysis was performed using wild-type cells (U87MG parentalcells), cells overexpressing the EGF receptor (A431 cells andUA87.wtEGFR cells) and UA87.Δ2-7 cells (data not shown). Similar bindingspecificity of mAb806 and ch806 was obtained using cells overexpressingEGFR and cells expressing the de2-7 EGFR. No binding was observed inwild-type cells. Scatchard analysis revealed a binding affinity forradiolabeled ch806 of 6.4×10⁹ M⁻¹ using U87MGde2-7 cells (data notshown).

Biodistribution analysis of the ch806 antibody was performed in BALB/cnude mice bearing U87MG-de2-7 xenograft tumors, and the results areshown in FIG. 34. Mice were injected with 5 μg of radiolabelled antibodyand were sacrificed in groups of four per time point at 8, 24, 48 and 74hours. Organs were collected, weighed and radioactivity measured in agamma counter. ¹²⁵I-labelled ch806 displays reduced targeting to thetumor compared to ¹¹¹In-labelled ch806, which has high tumor uptake andcumulative tumor retention over the 74 hour time period. At 74 hours,the ¹¹¹In-labelled antibody displays approximately 30% ID/gram tissueand a tumor to blood ratio of 4.0 (FIG. 35). The ¹¹¹In-labelled ch806shows some nonspecific retention in the liver, spleen and kidneys. Thisis common for the use of this isotope and decreases with time, whichsupports that this binding is non-specific to ch806 and due to ¹¹¹Inbinding.

Chimeric antibody ch806 was assessed for therapeutic efficacy in anestablished tumor model. 3×10⁶ U87MG.Δ2-7 cells in 100 μl of PBS wereinoculated s.c. into both flanks of 4-6 week old female nude mice(Animal Research Center, Western Australia, Australia). The mAb806 wasincluded as a positive control. The results are depicted in FIG. 36.Treatment was started when tumors had reached a mean volume of 50 mm³and consisted of 1 mg of ch806 or mAb806 given i.p. for a total of 5injections on the days indicated. Tumor volume in mm³ was determinedusing the formula (length×width²)/2, where length was the longest axisand width the measurement at right angles to the length. Data wasexpressed as mean tumor volume+/−S.E. for each treatment group. Thech806 and mAb806 displayed nearly identical anti-tumor activity againstU87MG.Δ2-7 xenografts.

Analysis of Ch806 Immune Effector Function Materials and Methods

Antibodies and Cell lines

Murine anti-de2-7 EGFR monoclonal mAb806, chimeric antibody ch806 (IgG₁)and control isotype matched chimeric anti-G250 monoclonal antibody cG250were prepared by the Biological Production Facility, Ludwig Institutefor Cancer Research, Melbourne, Australia. Both complement-dependantcytotoxicity (CDC) and antibody-dependent cellular-cytotoxicity (ADCC)assays utilized U87MG.de2-7 and A431 cells as target cells. Thepreviously described U87MG.de2-7 cell line is a human astrocytoma cellline infected with a retrovirus containing the de2-7EGFR (Nishikawa etal. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7727-31). Human squamouscarcinoma A431 cells were purchased from the American Type CultureCollection (Manassas, Va.). All cell lines were cultured in DMEM/F-12with Glutamax (Life Technologies, Melbourne, Australia) supplementedwith 10% heat-inactivated FCS (CSL, Melbourne, Australia), 100 units/mlpenicillin and 100 μg/ml streptomycin. To maintain selection forretrovirally transfected U87MG.de2-7 cells, 400 μg/ml G418 was includedin the media.

Preparation of Human Peripheral Blood Mononuclear Cells PBMC EffectorCells

PBMCs were isolated from healthy volunteer donor blood. Heparinizedwhole blood was fractionated by density centrifugation on Ficoll-Hypaque(ICN Biomedical Inc., Ohio, USA). PBMC fractions was collected andwashed three times with RPMI⁺ 1640 supplemented with 100 U/ml penicillinand 100 μg/ml streptomycin, 2 mM L-glutamine, containing 5%heat-inactivated FCS.

Preparation of Target Cells

CDC and ADCC assays were performed by a modification of a previouslypublished method (Nelson, D. L. et al. (1991) In: J. E. Colignan, A. M.Kruisbeek, D. D. Margulies, E. M. Shevach, and W. Strober (eds.),Current Protocols in Immunology, pp. 7.27.1. New York: Greene PublishingWiley Interscience). Briefly, 5×10⁶ target U87MG.de2-7 and A431 cellswere labeled with 50 μCi⁵¹Cr (Geneworks, Adelaide, Australia) per 1×10⁶cells and incubated for 2 hr at 37° C. The cells were then washed threetime with PBS (0.05M, pH 7.4) and a fourth wash with culture medium.Aliquots (1×10⁴ cells/50 μl) of the labeled cells were added to eachwell of 96-well microtitre plates (NUNC, Roskilde, Denmark).

CDC Assay

To 50 μl labeled target cells, 50 μl ch806 or isotype control antibodycG250 were added in triplicate over the concentration range 0.00315-10μg/ml, and incubated on ice 5 min. Fifty μl of freshly prepared healthydonor complement (serum) was then added to yield a 1:3 final dilution ofthe serum. The microtitre plates were incubated for 4 hr at 37° C.Following centrifugation, the released ⁵¹Cr in the supernatant wascounted (Cobra II automated Gamma Counter, Canberra Packard, Melbourne,Australia). Percentage specific lysis was calculated from theexperimental ⁵¹Cr release, the total (50 μl target cells+100 μl 10%Tween 20) and spontaneous (50 μl target cells+100 μl medium) release.

ADCC Assay

ch806-mediated ADCC effected by healthy donor PBMCs was measured by two4-hr ⁵¹1Cr release assays. In the first assay, labelled target cellswere plated with the effector cells in 96-well “U” bottom microplates(NUNC, Roskilde, Denmark) at effector/target (E:T) cell ratios of 50:1.For ADCC activity measurements, 0.00315-10 μg/ml (final concentration)test and control antibodies were added in triplicate to each well. Inthe second ADCC assay, the ADCC activity of ch806 was compared with theparental murine mAb806 over a range of Effector: Target cell ratios withthe test antibody concentration constant at 1 μg/ml. In both assays,micotitre plates were incubated at 37° C. for 4 hours, then 50 μlsupernatant was harvested from each well and released ⁵¹Cr wasdetermined by gamma counting (Cobra II automated Gamma Counter, CanberraPackard, Melbourne, Australia). Controls included in the assayscorrected for spontaneous release (medium alone) and total release (10%Tween20/PBS). Appropriate controls with the same subclass antibody wererun in parallel.

The percentage cell lysis (cytotoxicity) was calculated according to theformula:

${{Percentage}\mspace{14mu} {Cytotoxicity}} = {\frac{\begin{matrix}{{{Sample}\mspace{14mu} {Counts}} -} \\{{Spontaneous}{\mspace{11mu} \;}{Release}}\end{matrix}}{\begin{matrix}{{{Total}{\mspace{11mu} \;}{Release}} -} \\{{Spontaneous}{\mspace{11mu} \;}{Release}}\end{matrix}} \times 100}$

The percent (%) cytotoxicity was plotted versus concentration ofantibody (μg/ml).

Results

The results of the CDC analyses are presented in FIG. 37. Minimal CDCactivity was observed in the presence of up to 10 μg/ml ch806 with CDCcomparable to that observed with isotype control cG250.

ch806 mediated ADCC on target U87MG.de2-7 and A431 cells at E:T ratio of50:1 is presented in FIG. 38. Effective ch806 specific cytotoxicity wasdisplayed against target U87MG.de2-7 cells, but minimal ADCC wasmediated by ch806 on A431 cells. The levels of cytotoxicity achievedreflect the number of ch806 binding sites on the two cell populations.Target U87MG.de2-7 cells express ˜1×10⁶ de2-7EGFR which are specificallyrecognized by ch806, while only a subset of the 1×10⁶ wild-type EGFRmolecules expressed on A431 cells are recognized by ch806 (see aboveExamples).

Further ADCC analyses were performed to compare the ADCC mediated by 1μg/ml ch806 on target U87MG.de2-7 cells with that effected by 1 μg/mlparental murine mAb806. Results are presented in FIG. 39. Chimerizationof mAb806 has effected marked improvement of the ADCC achieved by theparental murine mAb with greater than 30% cytotoxicity effected at E:Tratios 25:1 and 50:1.

The lack of parental murine mAb806 immune effector function has beenmarkedly improved upon chimerization. ch806 mediates good ADCC, butminimal CDC activity.

EXAMPLE 20 Generation of Anti-Idiotype Antibodies to Chimeric Antibodych806

To assist the clinical evaluation of mAb806 or ch806, laboratory assaysare required to monitor the serum pharmacokinetics of the antibodies andquantitate any immune responses to the mouse-human chimeric antibody.Mouse monoclonal anti-idiotypic antibodies (anti-ids) were generated andcharacterized for suitability as ELISA reagents for measuring ch806 inpatient sera samples and use as positive controls in human anti-chimericantibody immune response analyses. These anti-idiotype antibodies mayalso be useful as therapeutic or prophylactic vaccines, generating anatural anti-EGFR antibody response in patients.

Methods for generating anti-idiotype antibodies are well known in theart (Chatterjee et al., 2001; Uemura et al., 1994; Steffens et al.,1997; Safa and Foon, 2001; Brown and Ling, 1988).

Briefly, mouse monoclonal anti-idiotypic antibodies (anti-ids) weregenerated as follows. Splenocytes from mice immunized with ch806 werefused with SP2/0-AG14 plasmacytoma cells and antibody producinghybridomas were selected through ELISA for specific binding to ch806 andcompetitive binding for antigen (FIG. 40). Twenty-five hybridomas wereinitially selected and four, designated LMH-11, -12, -13, and-14,secreted antibodies that demonstrated specific binding to ch806, mAb806and were able to neutralize ch806 or mAb806 antigen binding activity(FIG. 41). The recognition of the ch806/mAb806 idiotope or CDR regionwas demonstrated by lack of cross-reactivity with purified polyclonalhuman IgG.

In the absence of readily available recombinant antigen de2-7 EGFR toassist with the determination of ch806 in serum samples, the ability ofthe novel anti-idiotype ch806 antibodies to concurrently bind 806variable regions was exploited in the development of a sensitive,specific ELISA for measuring ch806 in clinical samples (FIG. 42). UsingLMH-12 for capture and Biotinylated-LMH-12 for detection, the validatedELISA demonstrated highly reproducible binding curves for measuringch806 (2 μg/ml-1.6 ng/ml) in sera with a 3 ng/ml limit of detection.(n=12; 1-100 ng/ml, Coefficient of Variation<25%; 100 ng/ml-5 μg/ml,Coefficient of Variation<15%). No background binding was evident withthe three healthy donor sera tested and negligible binding was observedwith isotype control hu3S193. The hybridoma produces high levels ofantibody LMH-12, and larger scale production is planned to enable themeasurement of ch806 and quantitation of any immune responses inclinical samples (Brown and Ling, 1988).

Results

Mice Immunization and hybridoma clone selection Immunoreactivity ofpre-and post-immunization sera samples indicated the development of hightiter mouse anti-ch806 and anti-huIgG mAbs. Twenty-five hybridomasproducing antibodies that bound ch806, but not huIgG, were initiallyselected. The binding characteristics of some of these hybridomas areshown in FIGS. 42A and 42B. Four of these anti-ch806 hybridomas withhigh affinity binding (clones 3E3, SB8, 9D6, and 4D8) were subsequentlypursued for clonal expansion from single cells by limiting dilution anddesignated Ludwig Institute for Cancer Research Melbourne Hybridoma(LMH)-11, -12, -13, and-14, respectively (FIG. 42).

Binding and Blocking Activities of Selected Anti-Idiotype Antibodies

The ability of anti-ch806 antibodies to concurrently bind two ch806antibodies is a desirable feature for their use as reagents in an ELISAfor determining serum ch806 levels. Clonal hybridomas, LMH-11, -12, -13,and-14 demonstrated concurrent binding (data not shown).

After clonal expansion, the hybridoma culture supernatants were examinedby ELISA for the ability to neutralize ch806 or mAb806 antigen bindingactivity with sEGFR621. Results demonstrated the antagonist activity ofanti-idiotype mAbs LMH-11, -12, -13, and-14 with the blocking insolution of both ch806 and murine mAb806 binding to plates coated withsEGFR (FIG. 41 for LMH-11, -12, -13).

Following larger scale culture in roller bottles the bindingspecificity's of the established clonal hybridomas, LMH-11, -12, -13,and-14 were verified by ELISA. LMH-11 through-14 antibodies wereidentified as isotype IgG1κ by mouse monoclonal antibody isotyping kit.

ch806 in Clinical Serum Samples Pharmacokinetic ELISA Assay Development

To assist with the determination of ch806 in serum samples, the abilityof the anti-idiotype ch806 antibodies to concurrently bind the 806variable region was exploited in the development of a sensitive andspecific ELISA assay for ch806 in clinical samples. The three purifiedclones LMH-11, -12, and-13 (FIGS. 49B and 49C, respectively werecompared for their ability to capture and then detect bound ch806 insera. Results indicated using LMH-12 (10 μg/ml) for capture andbiotinylated LMH-12 for detection yielded the highest sensitivity forch806 in serum (3 ng/ml) with negligible background binding.

Having established the optimal pharmacokinetic ELISA conditions using 1μg/ml anti-idiotype LMH-12 and 1 μg/ml biotinylated LMH-12 for captureand detection, respectively, validation of the method was performed.Three separate ELISAs were performed in quadruplicate to measure ch806in donor serum from three healthy donors or 1% BSA/media with isotypecontrol hu3 S 193. Results of the validation are presented in FIG. 43and demonstrate highly reproducible binding curves for measuring ch806(2 μg/ml-1.6 ng/ml) in sera with a 3 ng/ml limit of detection. (n=12;1-100 ng/ml, Coefficient of Variation<25%; 100 ng/ml-5 μg/ml,Coefficient of Variation<15%). No background binding was evident withany of the three sera tested and negligible binding was observed withisotype control hu3S193.

EXAMPLE 21 Assessment of Carbohydrate Structures and AntibodyRecognition

Experiments were undertaken to further assess the role of carbohydratestructures in the binding and recognition of the EGFR, both amplifiedand de2-7 EGFR, by the mAb806 antibody.

To determine if carbohydrate structures are directly involved in themAb806 epitope, the recombinant sEGFR expressed in CHO cells was treatedwith PNGase F to remove N-linked glycosylation. Following treatment, theprotein was run on SDS-PAGE, transferred to membrane and immunoblottedwith mAb806 (FIG. 44). As expected, the deglycosylated sEGFR ran fasteron SDS-PAGE, indicating that the carbohydrates had been successfullyremoved. The mAb806 antibody clearly bound the deglycosylated materialdemonstrating the antibody epitope is peptide in nature and not solely aglycosylation epitope.

Lysates, prepared from cell lines metabolically labelled with ³⁵S, wereimmunoprecipitated with different antibodies directed to the EGFR (FIG.45). As expected, the 528 antibody immunoprecipitated three bands fromU87MG.Δ2-7 cells, an upper band corresponding to the wild-type (wt) EGFRand two lower bands corresponding to the de2-7 EGFR. These two de2-7EGFR bands have been reported previously and are assumed to representdifferential glycosylation (Chu et al. (1997) Biochem. J. June 15; 324(Pt 3): 885-861). In contrast, mAb806 only immunoprecipitated the twode2-7 EGFR bands, with the wild-type receptor being completely absenteven after over-exposure (data not shown). Interestingly, mAb806 showedincreased relative reactivity with the lower de2-7 EGFR band butdecreased reactivity with the upper band when compared to the 528antibody. The SC-03 antibody, a commercial rabbit polyclonal antibodydirected to C-terminal domain of the EGFR, immunoprecipitated the threeEGFR bands as seen with the 528 antibody, although the total amount ofreceptor immunoprecipitated by this antibody was considerably less. Nobands were observed when using an irrelevant IgG2b antibody as a controlfor mAb806 (see Example 18).

The 528 antibody immunoprecipitated a single band from U87MG.wtEGFRcells corresponding to the wild-type receptor (FIG. 45). mAb806 alsoimmunoprecipitated a single band from these cells, however, this EGFRband clearly migrated faster than the 528 reactive receptor. The SC-03antibody immunoprecipitated both EGFR reactive bands from U87MG.wtEGFRcells, further confirming that the mAb806 and 528 recognize differentforms of the EGFR in whole cell lysates from these cells.

As observed with U87MG.wtEGFR cells, the 528 antibody immunoprecipitateda single EGFR band from A431 cells (FIG. 45). The 528 reactive EGFR bandis very broad on these low percentage gels (6%) and probably reflectsthe diversity of receptor glycosylation. A single EGFR band was alsoseen following immunoprecipitation with mAb806. While this EGFR band didnot migrate considerably faster than the 528 overall broad reactiveband, it was located at the leading edge of the broad 528 band in areproducible fashion. Unlike U87MG.Δ2-7 cell lysates, the total amountof EGFR immunoprecipitated by mAb806 from A431 lysates was considerablyless than with the 528 antibody, a result consistent with our Scatcharddata showing mAb806 only recognizes a portion of the EGFR on the surfaceof these cells (see Example 4). Immunoprecipitation with SC-03 resultedin a single broad EGFR band as for the 528 antibody. Similar resultswere obtained with FINS cells (data not shown). Taken together, thisdata indicates that mAb806 preferentially reacts with faster migratingspecies of the EGFR, which may represent differentially glycosylatedforms of the receptor.

In order to determine at what stage of receptor processing mAb806reactivity appeared a pulse/chase experiment was conducted. A431 andU87MG.Δ2-7 cells were pulsed for 5 min with ³⁵S methionine/cysteine,then incubated at 37° C. for various times before immunoprecipitationwith mAb806 or 528 (FIG. 46). The immunoprecipitation pattern in A431cells with the 528 antibody was typical for a conformational dependentantibody specific for the EGFR. A small amount of receptor wasimmunoprecipitated at 0 min (i.e. after 5 min pulse) with the amount oflabelled EGFR increasing at each time point. There was also a concurrentincrease in the molecular weight of the receptor with time. In contrast,the mAb806 reactive EGFR material was present at high levels at 0 min,peaked at 20 min and then reduced at each further time point. Thus, itappears that mAb806 preferentially recognizes a form of the EGFR foundat an early stage of processing.

The antibody reactivity observed in pulse-labelled U87MG.Δ2-7 cells wasmore complicated. Immunoprecipitation with the 528 antibody at 0 minrevealed that a small amount of the lower de2-7 EGFR band was labelled(FIG. 46). The amount of 528 reactive de2-7 EGFR lower band increasedwith time, peaking at 60 min and declining slowly at 2 and 4 h. Nosignificant amount of the labelled upper band of de2-7 EGFR was detecteduntil 60 min, after which the level continued to increase until the endof the time course. This clearly indicates that the upper de2-7 EGFR isa more mature form of the receptor. mAb806 reactivity also varied duringthe time course study, however mAb806 preferentially precipitated thelower band of the de27 EGFR. Indeed, there were no significant levels ofmAb806 upper band seen until 4 h after labeling.

The above experiments suggest that mAb806 preferentially reacts with amore immature glycosylation form of the de2-7 and wtEGFR. Thispossibility was tested by immunoprecipitating the EGFR from differentcells lines labelled overnight with ³⁵S methionine/cysteine and thensubjecting the resultant precipitates to Endoglycosidase H (Endo H)digestion. This enzyme preferentially removes high mannose typecarbohydrates (i.e. immature glycosylation) from proteins while leavingcomplex carbohydrates (i.e. mature glycosylation) intact.Immunoprecipitation and digestion with Endo H of labelled U87MG.Δ2-7cell lysates with 528, mAb806 and SC-03 gave similar results (FIG. 47).

As predicted, the lower de2-7 EGFR band was fully sensitive to Endo Hdigestion, migrating faster on SDS-PAGE after Endo H digestion,demonstrating that this band represents the high mannose form of thede2-7 EGFR. The upper de2-7 EGFR band was essentially resistant to EndoH digestion, showing only a very slight difference in migration afterEndo H digestion, indicating that the majority of the carbohydratestructures are of the complex type. The small but reproducible decreasein the molecular weight of the upper band following enzyme digestionsuggests that while the carbohydrates on the upper de2-7 EGFR band arepredominantly of the complex type, it does possess some high mannosestructures. Interestingly, these cells also express low amounts ofendogenous wtEGFR that is clearly visible following 528immunoprecipitation. There was also a small but noticeable reduction inmolecular weight of the wild-type receptor following Endo H digestion,indicating that it also contains high mannose structures.

The sensitivity of the immunoprecipitated wtEGFR to Endo H digestion wassimilar in both U87MG.wtEGFR and A431 cells (FIG. 47). The bulk of thematerial precipitated by the 528 antibody was resistant to the Endo Henzyme although a small amount of the material was of the high mannoseform. Once again there was a small decrease in the molecular weight ofthe wtEGFR following Endo H digestion suggesting that it does containsome high mannose structures. The results using the SC-03 antibody weresimilar to the 528 antibody. In contrast, the majority of the EGFRprecipitated by mAb806 was sensitive to Endo H in both U87MG.wtEGFR andA431 cells, confirming that mAb806 preferentially recognizes the highmannose form of the EGFR. Similar results were obtained with HN-5 cells,wherein the majority of the material precipitated by mAb806 wassensitive to Endo H digestion, while the majority of the materialprecipitated by mAb528 and SC-03 was resistant to Endo H digestion (datanot shown).

Cell surface iodination of the A431 cell line, was performed with ¹²⁵Ifollowed by immunoprecipitation with the 806 antibody. The protocol forsurface iodination was as follows: The cell lysis, immunoprecipitation,Endo H digestion, SDS PAGE and autoradiography are as described aboveherein. For labeling, cells were grown in media with 10% FCS, detachedwith EDTA, washed twice with PBS then resuspended in 400 μl of PBS(approx. 2-3×10⁶ cells). To this was added 15 μl of ¹²⁵I (100 mCi/mlstock), 100 μl bovine lactoperoxidase (1 mg/ml) stock 10 μl H₂O₂ (0.1%stock) and this was incubated for 5 min. A further 10 μl H₂O₂ was thenadded and the incubation continued for a further 3 min. Cells were thenwashed again 3 times with PBS and lysed in 1% Triton. Cell surfaceiodination of the A431 cell line with lactoperoxidase, followed byimmunoprecipitation with the 806 antibody, showed that, similar to thewhole cell lysates described above, the predominant form of the EGFRrecognized by 806 bound on the cell surface of A431 cells was sensitiveto EndoH digestion (FIG. 48). This confirms that the form of EGFR boundby 806 on the cell surface of A431 cells is an EndoH sensitive form andthus is the high mannose type.

EXAMPLE 22 Humanized (Veneered) Antibody 806

A. hu806 Construction

An expression vector for a humanized 806 antibody (hu806) wasconstructed. The vector, termed 8C65AAG (11891 bp; SEQ ID NO:41), wasdesigned to contain both genes for a full length hu806 in a single GSpromoter-driven gene expression cassette (FIGS. 53 and 54).

The heavy chain variable (VH) and constant (CH) regions (SEQ ID NOS:42and 43, respectively) are shown in FIG. 55A, with the VH region CDR1,CDR2, and CDR3 (SEQ ID NOS:44, 45, and 46, respectively) indicated byunderlining.

The light chain variable (VL) and constant (CL) regions (SEQ ID NOS:47and 48, respectively) are shown in FIG. 55B, with the VL region CDR1,CDR2, and CDR3 (SEQ ID NOS:49, 50, and 51, respectively) indicated byunderlining.

To obtain a humanized 806 antibody construct, the veneering (v)technology (Daugherty et al. (1991) Polymerase chain reactionfacilitates the cloning, CDR-grafting, and rapid expression of a murinemonoclonal antibody directed against the CD18 component of leukocyteintegrins. Nucleic Acids Res. 19(9), 2471-6; U.S. Pat. No. 6,797,492 toDaugherty; Padlan, E. A. (1991) A possible procedure for reducing theimmunogenicity of antibody variable domains while preserving theirligand-binding properties. Mol. Immunol. 28(4-5), 489-98; EuropeanPatent No. 519596 to Padlan et al.) was employed. In order to minimizethe immunogenicity of 806 antibody variable domains, while preservingligand-binding properties, replacement of the surface-exposed residuesin the framework regions which differ from those usually found in humanantibodies was undertaken. To achieve this, VL and VH chain of the mousemonoclonal antibody (mAb) 806 have been re-engineered by gene-synthesisand overlapping PCR primer technology. The CL (kappa) chain wasassembled in the same manner. To demonstrate the preservation of intactbinding sites, vVL and vVH were also expressed in a scFv format thatdemonstrated good binding to the synthetic peptide that comprises the806 antigenic epitope by ELISA and to recombinant EGF Receptor (EGFR)extracellular domain (ECD) as measured by surface plasmon resonance(SPR) analysis.

The v806VL and v806VH have been engineered into a full length human IgG1context using a codon-optimized kappa-LC and a newly designed codon-andsplice-site optimized human IgG1 heavy chain constant region to achievestable gene expression in NS0 and CHO cell systems. The expressionsystem is based on the LONZA GS expression system using the pEE12.4 andpEE6.4 heavy and light chain expression vectors as provided by LONZABiologics.

The hu806 antibody product (FIG. 55) obtained by transient expression ofthe 8C65AAG vector was reactive with recombinant EGFR-ECD by SPR, andwith the synthetic EGFR 806 peptide epitope by ELISA. The 8C65AAG vectorwas transferred to LICR Affiliate Christoph Renner (University ofZurich) for generation of stable GS-NS0 hu806 cell lines and to LICR,Melbourne Centre, for the generation of GS-CHO hu806 cell lines.

Strategy for construction, amplification and cloning of hu806 antibodygenes

Veneering and Codon Optimization

Antibody veneering is a humanization strategy aimed at countering HAMA(human anti-mouse antibody) responses. Mouse mAbs are considered“foreign” antigens by a patient's immune system and an immune responseis induced, even upon a single administration, preventing further use ofthe reagent in those patients. In the first step of the mAb806 veneeringprocess, the amino acid sequences of the VL and VH chains in mAb806 wereanalyzed, and each amino acid residue in the mAb806 protein sequence wasgraded for surface exposure (FIG. 56 and FIG. 57). Only those aminoacids that resided on the outside of the antibody molecule wereconsidered for possible modification, as these were the only ones thatwould be exposed to antibody recognition. Using BLAST, the mAb806protein sequence was compared to three human antibody sequences(VH36germ, CAD26810, and AAA37941). Wherever a mAb806 surface residuedid not match the consensus of the human antibody sequences, thatresidue was identified to be changed to the consensus sequence.Initially 12 amino acids in the VL were subjected to veneering; and 14in the VH chain of ch806 (FIG. 56 and FIG. 57).

Codon optimization is a means of improving the heterologous expressionof antibodies or other proteins based on the codon bias of the systemused to express these antibodies. One of the goals in the creation ofhu806 was to utilize codon optimization to improve expression levels forthis antibody. The expression system is based upon the LONZA GSexpression system using the pEE12.4 and pEE6.4 HC and LC expressionvectors as provided by LONZA Biologics and NS0 and/or CHO cells asproduction cells. Thus, decisions about which codon to use for a givenamino acid were made with consideration for whether or not that codonwould be favored in the NS0/CHO expression systems.

Construction and Amplification of 806 DNA Sequences by PCR

The sequences for veneered, codon optimized versions of the variableheavy (VH) and variable light (VL) regions of the hu806 antibody weresynthesized in the following manner: For each region (VH or VL), 8-10oligonucleotides were designed as overlapping sense and antisenseprimers. These oligos would overlap each other in such a way as to coverthe entire hu806 VH or VL sequence, including the signal sequence,coding sequences, introns, and include a HindIII site at the 5′ terminusand a 3′ BamHI site at the 3′ terminus. The oligonucleotide maps arepresented in FIGS. 56B and 57B, and the primer details are providedbelow.

Briefly, the hu806 VH or VL was assembled by PCR as follows: Initiallyv806hc- or v806lc-oligos 1, 2, 3, 4, oligos 5, 6, and oligos 7, 8, 9, 10were combined in three separate reactions. Aliquots (50 pmol) of eachflanking oligo, and 5 pmol of each internal oligo were added to a 50 μlPCR reaction containing 25 μl of 2× HotStar Taq Master Mix (Qiagen) and48 μl of nuclease free water. The thermo cycle program was as follows:95° C.; 15″, [94° C.; 30″, 58° C.; 30″, 72° C.; 30″]×20 cycles, 72° C.;10″, 4° C. The products of these three reactions were excised afterseparation by gel electrophoresis. They were then purified using a saltcolumn (Qiagen-Qiaspin Minipreps), and combined. These products werefurther amplified by PCR using primers 1 and 10. The product of thissecond reaction included restriction enzyme sites for HindIII and BamHI,enabling insertion into expression plasmids.

Oligonucleotides Used to PCR Synthesize the hu806 V-Regions:

SEQ ID NO: v806 VH: v806hc-1:  GAGAAGCTTGCCGCCACCATGGATTGGACCTGGCGCATTC52 v806hc-2: CCCTTCCTCCTCACTGGGATTTGGCAGCCCCTTACCTGTGGCGGCTGCTA 53CCAGAAAGAGAATGCGCCAGGTCCAATCC v806hc-3:CCCAGTGAGGAGGAAGGGATCGAAGGTCACCATCGAAGCCAGTCAAGG 54GGGCTTCCATCCACTCCTGTGTCTTCTCTAC v806hc-4:GACTCGGCTTGACAAGCCCAGGTCCACTCTCTTGGAGCTGCACCTGGCT 55GTGGACACCTGTAGAGAAGACACAGGAGTGG v806hc-5:GGGCTTGTCAAGCCGAGTCAAACTTTGTCCCTAACATGTACTGTGTCCG 56GATACTCTATCTCATCAGATTTTGCGTGGAATTGG v806hc-6:CCCAGAGTATGATATGTAGCCCATCCATTCTAAACCTTTCCCTGGTGGCT 57GCCTTATCCAATTCCACGCAAAATCTGATG v806hc-7:GGGCTACATATCATACTCTGGGAACACCAGATATCAACCCTCTCTGAAA 58AGCCGGATCACAATCACTAGGGACACGTCG v806hc-8:GCAGTAATATGTTGCTGTGTCTGGGGCTGTAACGGAGTTCAGCTGCAGG 59AAGAACTGGCTCTTCGACGTGTCCCTAGTGATTG v806hc-9:CCAGACACAGCAACATATTACTGCGTAACCGCTGGCAGAGGCTTCCCCT 60ATTGGGGACAGGGCACCCTAGTGACAGTGAGC v806hc-10:CACGGATCCATCTTACCGCTGCTCACTGTCACTAGGGTG 61 v806 VL: v806lc-1:GAGAAGCTTGCCGCCACCATGGATTG 62 v806lc 2:CTGGGATTTGGCAGCCCCTTACCTGTTGCGGCTGCTACAAGAAACAGTA 63TTCTCCAAGTCCAATCCATGGTGGCGGCAAG v806lc 3: GGGGCTGCCAAATCCCAGTGAGGAGGAAGGGATCGAAGGTGACCATCG 64AAGCCAGTCAAGGGGGCTTCCATCCACTCC v806lc 4: CATGCTGGATGGACTCTGAGTCATCTGAATATCACTGTGAACACCTGTA 65GAGAAGACACAGGAGTGGATGGAAGCCC v806lc 5: CTCAGAGTCCATCCAGCATGTCAGTCTCCGTGGGAGATAGGGTGACGAT 66AACCTGTCATTCAAGCCAAGACATCAACTCC v806lc 6: GTTCCGTGATAGATTAGTCCTTTGAAGGACTTACCAGGCTTCTGTTGGA 67GCCATCCAATATTGGAGTTGATGTCTTGGCTTG v806lc 7:CAAAGGACTAATCTATCACGGAACAAACTTGGACGACGGCGTGCCATC 68GAGATTTTCAGGGTCTGGCAGCGGGACCGACTATAC v806lc 8: GTGCTGGACGCAGTAGTATGTGGCAAAGTCTTCTGGCTCTAAGCTAGAG 69ATGGTCAGTGTATAGTCGGTCCCGCTG v806lc-9: CATACTACTGCGTCCAGCACGCTCAGTTCCCCTGGACATTCGGCGGCGG 70CACAAAACTGGAAATCAAACGTGAGTAGGG v806lc 10:  CTCGGATCCCTACTCACGTTTGATTTCC71hu806 CL:

A codon-optimized version of the constant kappa light chain (CL) wasprepared in a manner similar to that used for the variable regionsHowever, the initial PCR step involved the creation of only twopreliminary products using oligos VKlcons-1, 2, 3, 4; and 5, 6, 7, 8. Inaddition, the flanking restriction sites for this product were BamHI andNotI prior to plasmid insertion.

Oligonucleotides used to PCR synthesize the hu806 CL-regions: SEQ ID NO:VK1cons-1: GACGGATCCTTCTAAACTCTGAGGGGGTCGGATGACG 72 VK1cons-2:GGAGCTGCGACGGTTCCTGAGGAAAGAAGCAAACAGGAT 73GGTGTTTAAGTAACAATGGCCACGTCATCCGACCCCCTC VK1cons-3:GGAACCGTCGCAGCTCCCTCCGTGTTCATCTTCCCCCCA 74TCCGACGAGCAACTGAAGTCAGGCACAGCCTCCGTGGTG VK1cons-4:GTGCGTTGTCCACTTTCCACTGGACTTTGGCCTCTCTTG 75GGTAAAAGTTATTAAGGAGGCACACCACGGAGGCTGTGC VK1cons-5:GTGGAAAGTGGACAACGCACTACAGAGCGGGAACTCTCAGGA 76AAGCGTGACAGAGCAGGACTCAAAAGATTCAACATACAGCC VK1cons-6:CTTCACAGGCATATACCTTGTGCTTTTCATAATCAGCTTTTGAC 77AGTGTCAGGGTAGAAGATAGGCTGTATGTTGAATCTTTTGAGTC VK1cons-7:GCACAAGGTATATGCCTGTGAAGTAACTCATCAGGG 78ACTCAGCAGCCCTGTCACTAAAAGTTTTAATAGAG VK1cons-8:CCTGCGGCCGCTTATCAGCATTCGCCTCTATTAAAACTTTTGGT 79 GAGAGGGhu806 CH:

A synthetic, humanized version of the IgG1 constant heavy chain (CH)gene (SEQ ID NO:80) was purchased from GeneArt, Regensburg, Germany. Thegene was codon optimized for expression in CHO/NS0 cells. Details of thegene sequence, restriction sites, etc., are shown in FIG. 58.

Construction of Expression Plasmids

For transient transfection and preliminary testing, hu806 VH and VLsequences prepared in the manner described above were ligated intoexpression vectors containing generic constant regions. These vectors,provided by LICR Affiliate Christoph Renner (University of Zurich,Switzerland), were known as pEAK8 HC (which contained a generic CH), anda 33-xm-lc (which contained a generic CL). Vectors were digested usingBamHI and HindIII in the presence of CIP then hu806 VH and VL wereligated into the corresponding vectors. The resulting plasmids were usedto transform Top10 chemically competent E. coli (Invitrogen) accordingto the manufacturer's directions. Transformed E. Coli were plated onLB+Ampicillin plates, and resistant clones were screened by restrictiondigestion and PCR. In general, eight positive clones detected in thismanner would be isolated and further amplified. DNA purified from thesecolonies were analyzed by automated DNA sequencing.

Codon-optimized versions of the constant regions were added to theseconstructs by restriction enzyme-digestion and ligation using BamHI andNotI. These transformants were selected, sequenced, and analyzed asstated above. Prior to the full-length antibody chains being ligatedinto the Lonza GS system the BamHI site between the variable andconstant region sequences was destroyed, in one case, by digestion usingBamHI, fill-in using DNA Polymerase, and blunt-end ligation.

Restriction fragments containing hu806 (VH+CH) or hu806 (VL+CL) werethen digested with NotI followed by HindIII. These digestions weredesigned to create a blunt end at the NotI site, and thus were done inseries in the following manner: The plasmid was first digested withNotI. Fully digested (single-cut) plasmid was separated byelectrophoresis using a 1% agarose gel. This product was then excisedand purified on a salt column and filled-in using DNA Polymerase. Theproduct of this reaction was salt-column purified and then digested withHindIII. This product (˜1.3 Kb for hu806 (VH+CH), and ˜0.8 Kb for hu806(VL+CL) was then separated by gel electrophoresis, excised, andpurified.

Vectors pEE12.4 and pEE6.4 (Lonza Biologics plc, Slough, UK) were eachdigested on HindIII and PmlI. hu806 (VH+CH) was ligated to pEE12.4 tocreate pEE12.4-hu806H, and hu806 (VL+CL) was ligated to pEE6.4 to createpEE6.4-hu806L.

After screening, a combined, double gene Lonza plasmid was created tocontain both the hu806 heavy and light chain sequences. Briefly, thepEE12.4-hu806H and pEE6.4-hu806L vectors were digested with NotI andSalI restriction enzymes. The resultant fragments, which contained theGS transcription unit and hCMV-MIE promoter, followed by the hu806 Heavyor Light chain expression cassette, were isolated and ligated together.The resulting “combined” Lonza plasmid (Designated 8C65AAG) was used forsingle-plasmid transient transfections in a HEK 293 system and stabletransfections in NS0 and CHO systems. A plasmid map is shown in FIG. 53.

Modifications to Constructs

The complete sequence verified amino acid sequences of the veneeredhu806 Hc and hu806 Lc are shown in comparison to mAb806 in FIG. 59 andFIG. 60, respectively. Flanking the hu806 sequence within the appendicesare asterisks (*) indicating initial veneering changes and numbers (1-8)refer to the numbered modifications No.1 to No.8 described herein.

With regard to FIG. 60, the reference file (mAb806 LC) incorrectlyindicates Histidine (H), not the correct Tyrosine (Y) at position 91;the subject of modification #1. The original, uncorrected file sequenceis included in FIG. 60, to illustrate the necessary modification made tohu806 at position 91.

A number of modifications were made to the hu806 cDNA sequences afterthe initial construction and sequencing phase. The reasons for makingthese modifications included: introduction of 4 restriction enzyme sitesfor sequence modification purposes, to correct 2 amino acid errors inthe sequence introduced during PCR, to correct one amino acid errorarising from the initial mAb806 documentation, and to engineer 4additional amino acid changes to effect additional veneering variants.The following 8 stages of modifications were performed:

1. hu806 VL: CDR3 H91Y

The document from which the original oligonucleotides were createdincorrectly stated that there was a CAC (Histidine, H) at position 91 inthe CDR3 of the mAb806 VL sequence. Site-directed mutagenesis was usedto generate the correct sequence of TAC (Tyrosine, Y; PatentWO02/092771). The consequent change in the amino acid sequence at thisposition was from CVQHAQF (SEQ ID NO:84) to CVQYAQF (SEQ ID NO:85). Thefinal DNA and translated protein sequence in comparison to ch806 areshown in FIG. 61.

-   Sense primer for the histidine to tyrosine modification of the hu806    VL region (PDV1; 40 mer)

(SEQ ID NO: 86) 5′-CCACATACTACTGCGTCCAGTACGCTCAGTTCCCCTGGAC-3′

-   Antisense primer for the histidine to tyrosine modification of the    hu806 VL region (PDV2; 20 mer)

5′-CTGGACGCAGTAGTATGTGG-3′ (SEQ ID NO: 87)2. hu806 Heavy Chain: Addition of Restriction Sites DraIII and FseI

Restriction enzyme sites were added to the introns surrounding the hu806VH and VL regions. These restriction sites (unique in the pREN vectorsystem, LICR) were designed to ease the process of making modificationsto the expression cassettes. The hu806 VH sequence, not including theinitial signal region, could be removed or inserted by single-digestionon DraIII. In addition, FseI could be used, in concert with NotI (pRENsystem) or EcoRI (Lonza System) to cut out the constant region,fulfilling the function of BamHI from the original sequence.

These modifications were achieved using a two-step PCR process. Theproducts were then digested with HindIII and BglII. They were thenligated into pREN vectors containing codon-optimized constant regions,which had been digested on HindIII and BamHI. This re-ligation processdestroyed the BamHI site.

-   Sense primer for variable region upstream of first DraIII site (806    heavy chain DraIII Up; 26 mer)

(SEQ ID NO: 88) 5′-GAGAAGCTTGCCGCCACCATGGATTG-3′

-   Antisense primer incorporating DraIII site I (806 heavy chain DraIII    Down; 28 mer)

(SEQ ID NO: 89) 5′-CACTGGGTGACTGGCTTCGATGGTGACC-3′

-   Sense primer for the HC variable region between the two DraIII sites    (806 heavy chain DraIII-FseI Up; 49 mer)

(SEQ ID NO: 90) 5′-GGTCACCATCGAAGCCAGTCACCCAGTGAAGGGGGCTTCCATCCACT CC-3′

-   Antisense primer incorporating the DraIII site II, and the FseI site    (806 heavy chain DraIII-FseI Down; 44 mer)

(SEQ ID NO: 91) 5′-CCAAGATCTGGCCGGCCACGGTGTGCCATCTTACCGCTGCTCAC-3′3. hu806 Light Chain: Addition of Restriction Sites RsrII and PacI

For the hu806 light chain, the restriction sites added were RsrII,having the same function as DraIII in the heavy chain, and PacI, whichmatched the function of FseI.

-   Sense primer for variable region upstream of first RsrII site (806    light chain RsrII Up; 22 mer)

5′-GAGAAGCTTGCCGCCACCATGG-3′ (SEQ ID NO: 92)

-   Antisense primer incorporating RsrII site I (806 light chain RsrII    Down; 25 mer)

5′-CGGTCCGCCCCCTTGACTGGCTTCG-3′ (SEQ ID NO: 93)

-   Sense primer for the LC variable region between the two RsrII sites    (806 light chain RsrII-PacI Up; 45 mer)

(SEQ ID NO: 94) 5′-CGAAGCCAGTCAAGGGGGCGGACCGCTTCCATCCACTCCTGTGTC- 3′

-   Antisense primer incorporating the RsrII site II, and the PacI site    (806 light chain RsrII-PacI Down: 50 mer)

(SEQ ID NO: 95) 5′-CCAAGATCTTTAATTAACGGACCGCTACTCACGTTTGATTTCCAGTTTTG-3′4. hu806 VH: Reveneering P85A

The protein sequence for the parental mAb806 at VH amino acids 81-87 isSVTIEDT (SEQ ID NO:96). As part of the veneering process, isoleucine andglutamic acid at positions 84 and 85 were changed to alanine-proline toread SVTAPDT (SEQ ID NO:97; FIG. 56). Upon further analysis, it wasdecided that alanine might have been a better choice than proline inthis case. Site-directed mutagenesis was used to generate this secondarychange (SVTAADT, SEQ ID NO:98) using the primers listed below. Final DNAand translated protein sequences are presented in FIG. 62.

-   Sense primer (Fx3; 49 mer)

(SEQ ID NO: 99) 5′-CTGCAGCTGAACTCCGTTACAGCCGCAGACACAGCAACATATTACTG CG-3′

-   Antisense primer (Fx4; 49 mer)

(SEQ ID NO: 100) 5′-CGCAGTAATATGTTGCTGTGTCTGCGGCTGTAACGGAGTTCAGCTGCAG-3′5. hu806 VH: Additional Veneering

The hu806 heavy chain variable region sequence underwent three furthermutations following the initial veneering: T70S, S76N and Q81K. Thechange at position 76 from serine to asparagine represented a correctionback to the original sequence of mAb806 molecule. The additional changesin the framework were included because they represent residues that arenot found in mouse antibodies but are found in human antibodies.Accordingly, the protein sequence TRDTSKSQFFLQ (SEQ ID NO:101) wasveneered to SRDTSKNQFFLK (SEQ ID NO:102). Final DNA and translatedprotein sequences in comparison to mAb806 are presented in FIG. 62.

-   Sense Primer for HC variable region 5′ PCR fragment    (hu806HCfx2-5p-U; 49 mer)

(SEQ ID NO: 103) 5′-GGTCACCATCGAAGCCAGTCACCCAGTGAAGGGGGCTTCCATCCACTCC-3′

-   Antisense Primer for 5′ PCR fragment, incorporates first two changes    (hu806HCfx2-5p-D; 45 mer)

(SEQ ID NO: 104) 5′-GATTCTTCGACGTGTCCCTTGAGATTGTGATCCGGCTTTTCAGAG- 3′

-   Sense Primer for 3′ PCR fragment, incorporates all changes    (hu806HCfx2-3p-U; 55 mer)

(SEQ ID NO: 105) 5′-CAAGGGACACGTCGAAGAATCAGTTCTTCCTGAAACTGAACTCCGTTACAGCCGC-3′

-   Antisense Primer for HC variable region 3′ PCR fragment    (hu806HCfx2-3p-D; 44 mer)

(SEQ ID NO: 106) 5′-CCAAGATCTGGCCGGCCACGGTGTGCCATCTTACCGCTGCTCAC-3′6. hu806 VL: E79Q Veneering

This was the only post-construction VL veneering modification performed.At position 79 site directed mutagenesis was employed to correct thesequence SSLEPE (SEQ ID NO:107) to SSLQPE (SEQ ID NO:108). Final DNA andtranslated protein sequences in comparison to ch806 are presented inFIG. 61.

-   Sense Primer for LC variable region 5′ PCR fragment (hu806LC-5p-U;    45 mer)

(SEQ ID NO: 109) 5′-CGAAGCCAGTCAAGGGGGCGGACCGCTTCCATCCACTCCTGTGTC-3′

-   Antisense Primer for 5′ PCR fragment, incorporates intended mutation    (hu806LC-5p-D; 34 mer)

(SEQ ID NO: 110) 5′-CTCTGGTTGTAAGCTAGAGATGGTCAGTGTATAG-3′

-   Sense Prime for LC variable region 3′ PCR fragment incorporates    intended mutation (hu806LC-3p-U; 45 mer)

(SEQ ID NO: 111) 5′-CCATCTCTAGCTTACAACCAGAGGACTTTGCCACATACTACTGCG-3′

-   Antisense Primer for LC variable region 3′ PCR fragment    (hu806LC-3p-D; 50 mer)

(SEQ ID NO: 112) 5′-CCAAGATCTTTAATTAACGGACCGCTACTCACGTTTGATTTCCAGTTTTG-3′7. hu806 Light Chain: Kappa Constant Region Splice-Junction Modification

This point mutation was required to correct an error in the splicing ofthe codon-optimized version of the kappa constant region. Prior to thischange, the portion of the amino acid chain beginning with VYACEVTH (SEQID NO:113) and continuing to the end of the molecule would not have beenincluded in the final antibody (FIG. 60).

-   Sense primer for LC constant kappa 5′ PCR fragment (F1; 21 mer)

5′-GGCGGCACAAAACTGGAAATC-3′ (SEQ ID NO: 114)

-   Antisense primer for LC constant kappa 5′ PCR fragment, incorporates    correction (F2; 59 mer)

(SEQ ID NO: 115) 5′-GATGAGTTACTTCACAGGCATATACTTTGTGCTTTTCATAATCAGCTTTTGACAGTGTC-3′

-   Sense primer for LC constant kappa 3′ PCR fragment, incorporates    correction (F3; 26 mer)

5′-AGTATATGCCTGTGAAGTAACTCATC-3′ (SEQ ID NO: 116)

-   Antisense primer for LC constant kappa 3′ PCR fragment. (F4; 17 mer)

5′-GCCACGATGCGTCCGGC-3′ (SEQ ID NO: 117)8. hu806 VH: N60Q

In addition to the veneering changes made to antibody 806 in the initialstages of construction, Asparagine at position 60 in VH CDR2 was changedto Glutamine at this time. N-Glycosylation follows the scheme: N X S/T,where X is any amino acid. The amino acid sequence from position 60 wasN P S, which follows this scheme. However, it is infrequently the casethat proline (as in our example) or cysteine is found at the X positionfor N-glycosylation. It was of concern that inconsistent glycosylationcould lead to variations in the reactivity of the antibody. Thus,asparagine was removed, and replaced with its most closely related aminoacid, glutamine, removing any potential for this site to be glycosylated(FIG. 59 and FIG. 62).

Binding of Veneered hu806 Antibody 8C65AAG construct

Transient transfection of 293FT cells with the final plasmid 8C65AAG wasperformed to enable the preparation of small quantities of hu806 forinitial antigen binding verification. Culture supernatants from severalsmall-scale replicate transient transfections were pooled, concentratedand hu806 antibody was collected using a protein-A chromatography step.Approximately 1-2 μg of hu806 antibody was obtained as measured by aquantitative huIgG1 ELISA and the antibody was analyzed by Biacore forbinding to recombinant EGFR-ECD (FIG. 63). Bovine immunoglobulin fromthe cell culture medium co-purified with hu806 and represented the majorfraction of total IgG, limiting quantitative assessment of hu806binding.

Sequencing Primers

-   RenVecUPSTREAM: Sense primer, begins sequencing upstream of variable    region in peak8, and a33xm vectors.

5′-GCACTTGATGTAATTCTCCTTGG-3′ (SEQ ID NO: 118)

-   RenVecDwnstrmHC: Antisense primer begins sequencing downstream of    variable region on peak8 heavy-chain plasmid. Anneals within    non-codon-optimized HC constant region.

5′-GAAGTAGTCCTTGACCAGG-3′ (SEQ ID NO: 119)

-   RenVecDwnstrmLC: Antisense primer, begins sequencing downstream of    variable region on a33-xm-lc light-chain plasmid. Anneals within    non-codon-optimized LC constant region.

5′-GAAGATGAAGACAGATGGTGCAG-3′ (SEQ ID NO: 120)

-   Upstrm Lonza: Sense primer, begins sequencing upstream of variable    region in Lonza vectors pEE 12.4 and pEE 6.4. Cannot be used with    combined Lonza because this is a duplicate region in the combined    plasmid.

5′-CGGTGGAGGGCAGTGTAGTC-3′ (SEQ ID NO: 121)

-   Dnstrm 6-4: Antisense primer, begins sequencing downstream of    constant region in Lonza vector pEE 6.4

5′-GTGATGCTATTGCTTTATTTG-3′ (SEQ ID NO: 122)

-   Dnstrm 12-4: Antisense primer, begins sequencing downstream of    constant region in Lonza vector pEE12.4

5′-CATACCTACCAGTTCTGCGCC-3′ (SEQ ID NO: 123)

-   Cod-Opt LC const E: Sense primer, internal to the codon-optimized    light-chain v-kappa constant region

5′-CCATCCTGTTTGCTTCTTTCC-3′ (SEQ ID NO: 124)

-   Cod-Opt LC const F: Antisense primer, internal to the    codon-optimized light-chain v-kappa constant region (vk).

5′-GACAGGGCTGCTGAGTC-3′ (SEQ ID NO: 125)

-   806HCspec: Sense primer, internal and unique to the veneered version    of the 806 HC variable region.

5′-GTGCAGCTCCAAGAGAGTGGAC-3′ (SEQ ID NO: 126)

-   806LCspec: Sense primer, internal and unique to the veneered version    of the 806 LC variable region.

5′-CAGAGTCCATCCAGCATGTC-3′ (SEQ ID NO: 127)

-   A GenBank formatted text document of the sequence and annotations of    plasmid 8C65AAG encoding the IgG1 hu806 is set forth in FIG. 64.-   FIG. 53 was created using Vector NTI (Invitrogen).-   FIGS. 59-62 were created using Vector NTI AlignX.

Discussion

The veneering of the 806 anti-EGF receptor antibody involved mutation of14 amino acids in the VH (FIG. 59 and FIG. 62), and 12 changes to the VLchain (FIG. 60 and FIG. 61) with codon optimization as indicated forexpression in mammalian CHO or NS0 cells. The final double gene vector,designated 8C65AAG, has been sequence-verified, and the coding sequenceand translation checked. Binding to recombinant EGFR extracellulardomain was confirmed by Biacore analyses using transiently expressedhu806 product.

Stable single clones producing high levels of intact hu806 antibody havebeen selected in glutamine-free medium as recommended by LONZA. Stableclones have been gradually weaned off serum to obtain serum-freecultures.

B. In vitro and in vivo Characterization of hu806

The higher producing stable GS-CHO hu806 transfectants 14D8, 15B2 and40A10 and GS-NS0 hu806 transfectant 36 were progressed and small scalecultures instigated to enable preliminary hu806 product purification andcharacterization. Results indicated similar physicochemical properties.Accordingly a larger scale (15 L) stirred tank culture was undertakenfor the highest producing transfectant (GS-CHO hu806 40A10) and purifiedproduct underwent additional in vitro characterization and in vivotherapy studies in U87MG.de2-7 and A431 xenograft models.

Methodology and Results Production and Down Stream Processing: SmallScale

The shake flasks experiments were performed with E500 shake flasks witha 100 mL cell culture volume. FIG. 76 presents the cell viability andantibody productivity charts for the four transfectants during theculture. Product concentration was estimated by ELISA using the 806anti-idiotype antibody LMH-12 (Liu et al. (2003) Generation ofanti-idiotype antibodies for application in clinical immunotherapylaboratory analyses. Hybrid Hybridomics. 22(4), 219-28) as coatingantibody, and ch806 Clinical Lot: J06024 as standard. Material atharvest was centrifuged and supernatant was 0.2 μm filtered then theantibodies were affinity purified by Protein-A chromatography.

Large Scale

The CHO-K1SV transfectant cell line expressing hu806 candidate clone40A10 was cultured in a 15 L stirred tank bioreactor with glucose shotfeeding for 16 days using CD-CHO (Invitrogen)/25 μM L-Methioninesulfoximine (MSX; Sigma)/GS supplements (Sigma) as the base media. FIG.76C presents the cell growth and volumetric production in the 15 Lstirred tank bioreactor. Final yield was 14.7 L at 58 mg/L by ELISA.

Material at harvest was centrifuged and supernatant was 0.2 μm filteredthen concentrated to 2 L using 2×30K membranes in Pall Centrimateconcentrator. Aliquots (4×500 ml) were subsequently applied to a 250 mLProtein A column and eluted with 50 mM Citrate pH 4.5 containing 200 mMNaCl. Eluted antibody from the 4 runs was then pooled, concentrated anddialyzed into PBS, pH 7.4.

The hu806 products from the small and large scale cultures werequantified by OD A280 nm. The antibody samples recovered from rProtein-Awere assessed by Size Exclusion Chromatography (SEC) (small scale, FIG.77; large scale, FIG. 78), 4-20% Tris-Glycine SDS-PAGE under reduced andnon-reduced conditions (FIGS. 79-81), and Isoelectric Focusing wasperformed with an Amersham Multiphor II Electrophoresis system on anAmpholine PAG plate (pH 3.5-9.5) according to the manufacturer'sinstructions (FIG. 82).

The Protein-A affinity purified hu806 antibodies displayed symmetricalprotein peaks and identical SEC elution profiles to the ch806 clinicalreference material. The SDS-PAGE gel profiles were consistent with animmunoglobulin. The IEF pattern indicated three isoforms with pI rangingfrom 8.66 to 8.82 which was consistent with the calculated pI of 8.4 forthe protein sequence.

Binding Analyses FACS Analysis

The estimates of antibody concentration determined for each sample bythe OD A280 nm were utilised for FACS analyses with the adenocarcinomacell line A431 cells (containing EGFR gene amplification). We havepreviously observed that mAb806 bound approximately 10% of the ˜2×10⁶wtEGFR expressed on A431 tumor cells compared with the wtEGFR-specificmAb528 (Johns et al. (2002) Novel monoclonal antibody specific for thede2-7 epidermal growth factor receptor (EGFR) that also recognizes theEGFR expressed in cells containing amplification of the EGFR gene. Int.J. Cancer. 98(3), 398-408). Cells were stained with either one of thefour hu806 samples, an irrelevant IgG2b antibody, or positive controlch806; each were assessed at a concentration of 20 μg/ml. Control forsecondary antibody alone was also included [Goat anti hu-IgG (Fcspecific) FITC conjugated]. Composite FACS binding curves are presentedin FIG. 83 and demonstrate equivalent staining for all constructs.

The cell binding characteristics of hu806 40A10 sample produced by largescale culture was also assessed by FACS for binding A431 as well asU87MG.de2-7 glioma cells expressing the variant EGFRvIII receptor (Johnset al., 2002). Representative results of duplicate analyses arepresented in FIG. 84 and FIG. 85, respectively. Controls included anirrelevant IgG2b antibody (shaded histograms), ch806 or 528 (binds bothwild-type and de2-7 EGFR) as indicated.

The ch806 and the hu806 antibody demonstrated similar staining of theA431 and U87MG.de2-7 cell lines supporting our previous observationsthat mAb806 specifically recognized the de2-7 EGFR and a subset of theover-expressed EGFR (Luwor et al. (2001) Monoclonal antibody 806inhibits the growth of tumor xenografts expressing either the de2-7 oramplified epidermal growth factor receptor (EGFR) but not wild-typeEGFR. Cancer Res. 61(14), 5355-61). As expected, the 528 antibodystained both the U87MG.de2-7 and A431 cell lines (FIGS. 84 and 85).

Cell Binding Analyses

The antigen binding capabilities of the radioimmunoconjugates wereassessed by cell adsorption assays (Lindmo et al. (1984) Determinationof the immunoreactive fraction of radiolabeled monoclonal antibodies bylinear extrapolation to binding at infinite antigen excess. J. Immunol.Methods. 72(1), 77-89) using the U87MG.de2-7 glioma cell line and A431epidermoid carcinoma cells expressing the amplified EGFR gene.

Immunoreactive fractions of hu806 and ch806 radioconjugates weredetermined by binding to antigen expressing cells in the presence ofexcess antigen. Results for U87MG.de2-7 cell binding of ¹²⁵I-hu806 and¹²⁵I-ch806 are presented in FIG. 86A over the cell concentration range20×10⁶ to 0.03×10⁶ cells/sample. Results for A431 cell binding of¹²⁵I-hu806 and ¹²⁵I-ch806 are presented in FIG. 86B over the cellconcentration range 200×10⁶ to 0.39×10⁶ cells/sample.

Scatchard analyses were used to calculate the association constant (Ka)(Lindmo et al., 1984). The binding of low levels (20 ng) of labeledantibody alone was compared with binding in the presence of excessunlabeled antibody. The immunoreactive fraction was taken into accountin calculating the amount of free, reactive antibody as previouslydescribed (Clarke et al. (2000) In vivo biodistribution of a humanizedanti-Lewis Y monoclonal antibody (hu3S193) in MCF-7 xenografted BALB/cnude mice. Cancer Res. 60(17), 4804-11) and specific binding (nM; totalantibody×% bound) was graphed against specific binding/reactive free(FIGS. 87 and 88). The association constant was determined from thenegative slope of the line.

The binding affinity for ¹²⁵I-hu806 binding EGFRvIII on U87MG.de2-7cells was determined to be 1.18×10⁹ M⁻¹. The Ka for ¹²⁵I-ch806 was1.06×10⁹ M⁻¹. These observations are in agreement with the reportedresults of Ka values for ¹¹¹In- and ¹²⁵I-ch806 of 1.36×10⁹ M-1 and1.90×10⁹ M⁻¹, respectively, which is highly comparable to that of theparental murine mAb806 of 1.1×10⁹ M⁻¹ (Panousis et al. (2005)Engineering and characterization of chimeric monoclonal antibody 806(ch806) for targeted immunotherapy of tumours expressing de2-7 EGFR oramplified EGFR. Br. J. Cancer. 92(6), 1069-77).

The scatchard analysis on A431 cells demonstrated high affinity bindingby both 806 constructs to a minor population of EGFR on these cells. TheKa for ¹²⁵I-ch806 was 0.61×10⁹ M⁻¹; and for ¹²⁵I-hu806 the Ka=0.28×10⁹M⁻¹.

Biosensor Analysis

Biosensor analyses were performed on a BIAcore 2000 biosensor using acarboxymethyldextran-coated sensor chip (CM5). The chip was derivatizedon channel 3 with the 806 epitope peptide (EGFR amino acids 287-302; SEQID NO:14; see U.S. patent application Ser. No. 11/060,646, filed Feb.17, 2005; U.S. Provisional Patent Application No. 60/546,602, filed Feb.20, 2004; and U.S. Provisional Patent Application No. 60/584,623, filedJul. 1, 2004, the disclosure of each is which is hereby incorporated inits entirety), using standard amine coupling chemistry. Channel 2 wasderivatized with a control antigen used for system suitabilitydetermination. Channel 1 was derivatized with ethanolamine and used as ablank control channel for correction of refractive index effects.Samples of hu806 were diluted in HBS buffer (10 mM HEPES, pH 7.4; 150 mMNaCl; 3.4 mM di-Na-EDTA; 0.005% Tween-20), and aliquots (120 μl)containing 50 nM, 100 nM, 150 nM, 200 nM, 250 nM and 300 nM wereinjected over the sensor chip surface at a flow rate of 30 μl/min. Afterthe injection phase, dissociation was monitored by flowing HBS bufferover the chip surface for 600 s. Bound antibody was eluted and the chipsurface regenerated between samples by injection of 20 μl of 10 mMsodium hydroxide solution. Positive control, ch806, was included. Thebinding parameters were determined using the equilibrium binding modelof the BIAevaluation software. FIG. 89 present the sensorgramsgenerated.

Dose dependant binding was observed with both hu806 and the positivecontrol, ch806, on channel 3. System suitability was confirmed by dosedependant binding of the appropriate monoclonal antibody to controlchannel 2. No cross reactivity was observed between hu806 (or ch806) andthe control antibody. Our analyses determined that the apparent K_(D)(1/Ka) was 37 nM for hu806 and 94 nM for ch806.

Antibody Dependent Cellular Cytotoxicity Analyses

ADCC analyses were performed using purified hu806 antibody 40A10preparation with target A431 adenocarcinoma cells and freshly isolatedhealthy donor peripheral blood mononuclear effector cells. Briefly, allanalyses were performed in triplicate with 1) 1 μg/ml each antibody overa range of effector to target cell ratios (E:T=0.78:1 to 100:1) and also2) at E:T=50:1 over a concentration range of each antibody (3.15ng/ml-10 μg/ml). Controls for antibody isotype, spontaneous and totalcytotoxicity were included in triplicate and calculations for specificcytotoxicity were as previously described (Panousis et al., 2005).Results are presented in FIG. 90.

The hu806 consistently demonstrated superior ADCC activity to thechimeric ch806 IgG1. In the representative experiment shown, hu806 at 1μg/mL effected an ADCC of 30% cytotoxicity in contrast to ch806 5%cytoxicity.

In vivo 806 Therapy Study

The therapeutic efficacy of hu806 was investigated using establishedA431 adenocarcinoma or U87MG-de2-7 glioma xenografts in BALB/c nudemice. To establish xenografts, mice were injected subcutaneously intothe right and left inguinal mammary line with 1×10⁶ A431 adenocarcinomacells or 1×10⁶ U87MG.de2-7 glioma cells in 100 μl of PBS. Tumor volume(TV) was calculated by the formula [(length×width²)/2] where length wasthe longest axis and width the measurement at right angles to length. Inan initial experiment, groups of five BALB/c nude mice (n=10 tumours/group) with established A431 or U87MG.de2-7 xenografts receivedtreatment of 1 mg hu806, or 1 mg ch806 antibody or PBS vehicle controlby IP injection. Therapy was administered on days 6, 8, 11, 13, 15 and18 for A431, and days 4, 6, 8, 11, 13 and 15 for the U87MG.de2-7 celllines respectively. Mean±SEM tumor volumes until termination of theexperiments due to ethical considerations of tumor burden are presentedin FIG. 91 for the A431 xenograft until day 25, and in FIG. 92 forU87MG.de2-7 xenografts until day 31.

The in vivo therapy assessments with hu806 showed a marked reduction inA431 xenograft growth compared with PBS vehicle control. The A431xenograft growth curve observed for hu806 was highly comparable to thech806 treatment group. In the established U87MG.de2-7 xenografts, thePBS control group was euthanized at day 20. The hu806 therapydemonstrated significant reduction in tumor growth by day 20 compared tothe PBS controls (P<0.001), and continued tumor growth retardation afterday 20 similar to the ch806 group.

Discussion

The Protein-A affinity purified hu806 antibodies displayed identical SECelution profiles to the ch806 clinical reference material, and SDS-PAGEgel profiles consistent with an immunoglobulin. The IEF pattern wasconsistent with the anticipated pI of 8.4.

Through Scatchard cell binding and Biosensor epitope binding analysesthe hu806 antibody demonstrated highly comparable binding curves andaffinity parameters to the ch806 antibody. The binding affinity of hu806and ch806 to EGFRvIII and over expressed wild-type EGFR are similar andin the low nanomolar range. Cell binding through FACS analyses supportedthese observations.

Furthermore, the hu806 demonstrates markedly improved ADCC over thech806 construct on target antigen positive A431 cells.

The in vivo therapeutic assessments with hu806 showed a marked reductionin A431 xenograft growth, which was highly comparable to the ch806treatment group. In the established U87MG.de2-7 xenografts, hu806therapy demonstrated significant reduction in tumor growth by day 20compared to the PBS controls and continued tumor growth retardationafter day 20 similar to the ch806 group.

EXAMPLE 23 Monoclonal Antibody 175

As discussed in Example 1, clone 175 (IgG2a) was selected for furthercharacterization.

a. Materials and Methods

Cell Lines

The Δ2-7EGFR transfected U87MG.Δ2-7(Huang et al. (1997) J. Biol. Chem.272, 2927-2935) and the A431 cell lines (Ullrich et al. (1984) Nature.309, 418-425) have been described previously. The hormone-independentprostate cell line DU145 (Mickey et al. (1977) Cancer Res. 37,4049-4058) was obtained from the ATCC (atcc.org).

All cell lines were maintained in DMEM (Life Technologies, Grand Island,N.Y.) containing 10% FCS (CSL, Melbourne), 2 mM glutamine (SigmaChemical Co, St. Louis), and penicillin/streptomycin (Life Technologies,Grand Island). In addition, the U87MG.Δ2-7 cell line was maintained in400 mg/ml of Geneticin (Life Technologies, Inc, Grand Island). BaF/3(Palacios et al. (1984) Nature. 309, 126-131) and BaF/3 cell linesexpressing different EGF receptors (Walker et al. (2004) J. Biol. Chem.2(79), 22387-22398) were maintained routinely in RPMI 1640 (GIBCO BRL)supplemented with 10% fetal calf serum (GIBCO BRL) and 10% WEHI-3Bconditioned medium(Ymer et al. (1985) Nature. 19-25; 317, 255-258) as asource of IL-3. All cell lines were grown at 37° C. in an air/CO₂(95%-5%) atmosphere.

Antibodies and Peptides

mAb806 and mAb175 were generated at the Ludwig Institute for CancerResearch (LICR) New York Branch and were produced and purified in theBiological Production Facility (Ludwig Institute for Cancer Research,Melbourne). The murine fibroblast line NR6_(ΔEGFR) was used asimmunogen. Mouse hybridomas were generated by immunizing BALB/c micefive times subcutaneously at 2- to 3-week intervals, with 5×10⁵-2×10⁶cells in adjuvant. Complete Freund's adjuvant was used for the firstinjection. Thereafter, incomplete Freund's adjuvant (Difco) was used.Spleen cells from immunized mice were fused with mouse myeloma cell lineSP2/0. Supernatants of newly generated clones were screened inhemadsorption assays for reactivity with cell line NR6, NR6_(wtEGFR),and NR6_(ΔEGFR) and then analyzed by hemadsorption assays with humanglioblastoma cell lines U87MG, U87MG_(wtEGFR). and U87MG_(ΔEGFR).

Intact mAbs (50 mg) were digested in PBS with activated papain for 2-3hours at 37° C. at a ratio of 1:20 and the papain was inactivated withiodoacetamide. The digestion was then passed over a column of Protein-Asepharose (Amersham) in 20 mM sodium phosphate buffer pH 8.0, with theflow-through further purified by cation exchange using on a Mono-Scolumn (Amersham). Protein was then concentrated using a 10,000 MWCOcentrifugal concentrator (Millipore). For Fab-peptide complexes a molarexcess of lyophilized peptide was added directly to the Fab andincubated for 2 hours at 4° C. before setting up crystallization trials.

Mapping of mAb 175 Using EGFR Fragments Expressed in Mammalian Cells

The day prior to transfection with these fragments, human 293Tembryonic-kidney fibroblasts were seeded at 8×10⁵ per well in 6-welltissue culture plates containing 2 ml of media. Cells were transfectedwith 3-4 μg of plasmid DNA complexed with Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. 24 to 48 hafter transfection, cell cultures were aspirated and cell mono layerslysed in 250μ; of lysis buffer (1% Triton X-100, 10% glycerol, 150 mMNaCl, 50 mM HEPES pH 7.4, 1 mM EGTA and Complete Protease Inhibitor mix(Roche). Aliquots of cell lysate (10-15 μl) were mixed with SDS samplebuffer containing 1.5% β-mercaptoethanol, denatured by heating for 5 minat 100° C. and electrophoresed on 10% NuPAGE Bis-Tris polyacrylamidegels (Invitrogen). Samples were then electro-transferred tonitrocellulose membranes that were rinsed in TBST buffer (10 mMTris-HCI, pH 8.0, 100 mM NaCl and 0.1% Tween-20) and blocked in TBSTcontaining 2.5% skim milk for 30 min at room temperature. Membranes wereincubated overnight at 4° C. with 0.5 μg/ml of mAb 175 in blockingbuffer. Parallel membranes were probed overnight with mAb 9B11 (1:5000,Cell Signaling Technology, Danvers, Mass.) to detect the c-myc epitope.Membranes were washed in TBST, and incubated in blocking buffercontaining horseradish peroxidase-conjugated rabbit anti-mouse IgG(Biorad) at a 1:5000 dilution for 2 h at room temperature. Blots werethen washed in TBST, and developed using autoradiographic film followingincubation with Western Pico Chemiluminescent Substrate (Pierce,Rockford, Ill.).

Mapping of mAb 175 Using EGFR Fragments Expressed in Mammalian Cells andYeast

A series of overlapping c-myc-tagged EGFR ectodomain fragments, startingat residues 274, 282, 290 and 298 and all terminating at amino acid 501and fused to growth hormone have been described previously(Johns et al.(2004) J. Biol. Chem. 279, 30375-30384). Expression of EGFR proteins onthe yeast cell surface was performed as previously described (Johns etal., 2004).

Briefly, transformed colonies were grown at 30° C. in minimal mediacontaining yeast nitrogen base, casein hydrolysate, dextrose, andphosphate buffer pH 7.4, on a shaking platform for approximately one dayuntil an OD₆₀₀ of 5-6 was reached. Yeast cells were then induced forprotein display by transferring to minimal media containing galactose,and incubated with shaking at 30° C. for 24 h. Cultures were then storedat 4° C. until analysis. Raw ascites fluid containing the c-mycmonoclonal antibody 9E10 was obtained from Covance (Richmond, Calif.).1×10⁶ yeast cells were washed with ice-cold FACS buffer (PBS containing1 mg/ml BSA) and incubated with either anti-c-myc ascites (1:50dilution), or human EGFR monoclonal antibody (10 μg/ml) in a finalvolume of 50 μl, for 1 hr at 4° C. The cells were then washed with icecold FACS buffer and incubated with phycoerythrin-labelled anti-mouseIgG (1:25 dilution), in a final volume of 50 μl for 1 h at 4° C.,protected from light. After washing the yeast cells with ice-cold FACSbuffer, fluorescence data was obtained with a Coulter Epics XL flowcytometer (Beckman-Coulter), and analyzed with WinMDI cytometry software(J. Trotter, Scripps University). For determination of linear versusconformational epitopes, yeast cells were heated at 80° C. for 30 min,then chilled on ice 20 min prior to labeling with antibodies. The seriesof EGFR mutants listed in Table 7 have been described previously (Johnset al., 2004).

Surface Plasmon Resonance (BIAcore)

A BIAcore 3000 was used for all experiments. The peptides containing theputative mAb806 epitope were immobilized on a CM5 sensor chip usingamine, thiol or Pms coupling at a flow rate of 5 μl/min (Wade et al.(2006) Anal. Biochem. 348, 315-317). The mAb806 and mAb 175 were passedover the sensor surface at a flow rate of 5 μl/min at 25° C. Thesurfaces were regenerated between runs by injecting 10 mM HCI at a flowrate of 10 μl/min.

Immunoprecipitation and Western Blotting

Cells were lysed with lysis buffer (1% Triton X-IOO, 30 mM HEPES, 150 mMNaCl, 500 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 150 nM aprotinin,1 mM E-64 protease inhibitor, 0.5 mM EDTA, and 1 mM leupeptin, pH 7.4)for 20 minutes, clarified by centrifugation at 14,000×g for 30 minutes,immunoprecipitated with the relevant antibodies at a final concentrationof 5 μg/ml for 60 minutes and captured by Sepharose-A beads overnight.Samples were then eluted with 2× NuPAGE SDS Sample Buffer (Invitrogen),resolved on NuPAGE gels (either 3-8% or 4-12%), electro-transferred ontoImmobilon-P transfer membrane (Millipore) then probed with the relevantantibodies before detection by chemoluminescence radiography.

Immunohistochemistry

Frozen sections were stained with 5 μg/ml mAb175 or irrelevant isotypecontrol for 60 min at room temperature. Bound antibody was detectedusing the Dako Envision+HRP detection system as per manufacturer'sinstructions. Sections were finally rinsed with water, counterstainedwith hematoxylin and mounted.

Xenograft Models

U87MG.Δ2-7 cells (3×10⁶) in 100 μL of PBS were inoculated s.c. into bothflanks of 4- to 6-week-old, female Balb/c nude mice (Animal ResearchCentre, Perth, Australia). All studies were conducted using establishedtumor models as reported previously (Perera et al. (2005) Clin. CancerRes. 11, 6390-6399). Treatment commenced once tumors had reached themean volume indicated in the appropriate figure legend. Tumor volume inmm was determined using the formula (length×width²)/2, where length wasthe longest axis and width was the perpendicular measurement. Data areexpressed as mean tumor volume±SE for each treatment group. All data wasanalyzed for significance by one-sided Student's t test where p<0.05 wasconsidered statistically significant. This research project was approvedby the Animal Ethics Committee of the Austin Hospital.

Generation and Characterization of Stable Cell Lines Expressing EGFRMutant Constructs

Mutations of the wtEGFR were generated using a site-directed mutagenesiskit (Stratagene, La Jolla, Calif.). The template for each mutagenesiswas the human EGFR cDNA (accession number x00588) (Ullrich et al. (1984)Nature. 309, 418-425). Automated nucleotide sequencing of each constructwas performed to confirm the integrity of the EGFR mutations. Wild-typeand mutant (C173A/C281A) EGFR were transfected into BaF/3 cells byelectroporation.

Stable cell lines expressing the mutant EGFR were obtained by selectionin neomycin-containing medium. After final selection, mRNA was isolatedfrom each cell line, reverse transcribed and the EGFR sequence amplifiedby PCR. All mutations in the expressed EGFR were confirmed by sequencingthe PCR products. The level of EGFR expression was determined by FACSanalysis on a FACStar (Becton and Dickinson, Franklin Lakes, N.J.) usingthe anti-EGFR antibody mAb528 (Masui et al. (1984) Cancer Res. 44,1002-1007; Gill et al. (1984) J. Biol. Chem. 259, 7755-7760) at 10 μg/mlin PBS, 5% FCS, 5 mM EDTA followed by Alexa 488-labeled anti-mouse Ig(1:400 final dilution). Background fluorescence was determined byincubating the cells with an irrelevant, class-matched primary antibody.All cells were routinely passaged in RPMI, 10% FCS, 10% WEHI3Bconditioned medium and 1.5 mg/ml G418.

EGF-Dependent Activation of Mutant EGFR

Cells expressing the wtEGFR or C271A/C283A-EGFR were washed andincubated for 3 hr in medium without serum or IL-3. Cells were collectedby centrifugation and resuspended in medium containing EGF (100 ng/ml)or an equivalent volume of PBS. Cells were harvested after 15 min,pelleted and lysed directly in SDS/PAGE sample buffer containingp-mercaptoethanol. Samples were separated on NUPAGE 4-12% gradient gels,transferred to Immobilon PVDF membrane and probed withanti-phosphotyrosine (4G10, Upstate Biotechnologies) or anti-EGFRantibodies (mAb806, produced at the LICR). Reactive bands were detectedusing chemiluminescence.

Effect of EGF and Antibodies on Cell Proliferation

Cells growing in log phase were harvested and washed twice with PBS toremove residual IL-3. Cells were resuspended in RPMI 1640 plus 10% FCSand seeded into 96-well plates at 10⁵ cells/well with carrier only orwith increasing concentrations of EGF. Where appropriate, a fixedconcentration of mAb528 or mAb806 (2 μg/well) was also added to thecultures. Proliferation was determined using the MTT assay (van deLoosdrecht et al. (1994) J. Immunol. Methods. 174, 311-320).

Reactivity with Conformation-Specific Antibodies

Cells were collected by centrifugation and stained with the control ortest antibodies (all at 10 μg/ml in FACS buffer for 40 min on ice,washed in FACS buffer) followed by Alexa 488-labeled anti-mouse Ig(1:400 final dilution, 20 min on ice). The cells were washed withice-cold F ACS buffer, collected by centrifugation, and analyzed on aFACScan; peak fluorescence channel and median fluorescence weredetermined for each sample using the statistical tool in Cell Quest(Becton and Dickinson). Background (negative control) fluorescence wasdeducted from all measurements. The median fluorescence values werechosen as most representative of peak shape and fluorescence intensityand were used to derive the ratio of mAb806 to mAb528 binding.

Crystal Structure Determinations of Fab 175, and Fab 806, Fab-PeptideComplexes and the NMR Structure of the 806 Peptide Epitope in Solution

Structures were determined by molecular replacement and refinementconverged with R=0.225/Rfree=0.289 for Fab806 and R=0.226/Rfree=0.279for Fab806:peptide; R=0.210/Rfree=0.305 for Fab806 andR=0.203/Rfree=0.257 for Fab806:peptide.

Crystals of native 806 Fab were grown by hanging drop vapor diffusionusing 10 mg/ml Fab and a reservoir containing 0.1M Sodium acetate bufferpH 4.6,6-8% PEG6000 and 15-20% Isopropanol. For data collection crystalswere transferred to a cryoprotectant solution containing 0.1M Sodiumacetate buffer pH 4.6, 10% PEG6000, 15-20% Isopropanol and 10% glycerol.Crystals were then mounted in a nylon loop and flash frozen directlyinto liquid nitrogen.

Crystals of 806 Fab-peptide complex were grown by hanging drop vapordiffusion using 10 mg/ml Fab-peptide complex and a reservoir containing0.2M ammonium acetate 16-18% PEG 5,000 monomethylether, crystals qualitywas then improved through seeding techniques. For data collectioncrystals were transferred to a cryoprotectant solution consisting ofreservoir supplemented with 25% glycerol. Crystals were then mounted ina nylon loop and flash frozen directly into liquid nitrogen.

Crystals of 175 Fab-peptide complex were initially grown by freeinterface diffusion using a Topaz crystallization system (Fluidigm, SanFrancisco). Microcrystals were grown by hanging drop vapor diffusionusing 7 mg/ml Fab with similar conditions 0.1M Bis-tris propane buffer,0.2M ammonium acetate and 18% PEG 10,000. Microcrystals were thenimproved by streak seeding into 0.15 m Sodium formate and 15% PEG 1500to yield small plate shaped crystals. For data collection crystals weretransferred to a cryoprotectant solution consisting of reservoirsupplemented with 25% glycerol. Crystals were then mounted in a nylonloop and flash frozen directly into liquid nitrogen.

Diffraction data on 806 Fab and 175 Fab complex crystals were collectedin-house using a R-AXIS IV detector on a Rigaku micromax-007 generatorfitted with AXCO optics, these data were then processed usingCrystalClear. 806 Fab-peptide complex data were collected on an ADSCquantum315 CCD detector at beamline X29, Brookhaven National Laboratory,these data were processed with HKL2000 (Otwinowski, Z. and Minor, W.(1997) Processing of X-ray diffraction data collected in oscillationmode. Academic Press (New York)) (data collection statistics are shownin Table 9). Native 806 Fab was solved by molecular replacement usingthe program MOLREP (Vagin, A. and Teplyakov, A. (1997) J. Appl. Cryst.30, 1022-1025) using the coordinates of the Fab structure 2E8 refinementof the structure was performed in REFMAC5 (Murshudov et al. (1997) Actacrystallographica 53, 240-255) and model building in Coot (Emsley, P.and Cowtan, K. (2004) Acta crystallographica 60, 2126-2132).

Both 806-peptide and 175 Fab-peptide structures were solved by molecularreplacement using the program MOLREP using the coordinates of the 806Fab structure, refinement and rebuilding were again performed inREFMAC5, and COOT and O. Validation of the final structures wereperformed with PROCHECK (Laskowski et al. (1993) J. Appl. Cryst. 26,283-291) and WHATCHECK (Hooft et al. (1996) Nature 381, 272).

NMR Studies

For NMR studies, ¹⁵N-Iabelled peptide was produced recombinantly as afusion to the SH2 domain of SHP2 using the method previously describedby Fairlie et al. (Fairlie et al. (2002) Protein expression andpurification 26, 171-178) except that the E. coli were grown inNeidhardt's minimal medium supplemented with ¹⁵NH₄Cl (Neidhardt et al.(1974) Journal of bacteriology 119, 736-747). The peptide was cleavedfrom the fusion partner using CNBr, purified by reversed-phase HPLC andits identity confirmed by MALDI-TOF mass spectrometry and N-terminalsequencing. The methionine residue within the 806 antibody-bindingsequence was mutated to leucine to enable cleavage from the fusionpartner, but not within the peptide itself.

Samples used for NMR studies were prepared in H_(2O) solution containing5% ²H₂O, 70 mM NaCl and 50 mM NaP0₄ at pH 6.8. All spectra were acquiredat 298K on a Bruker Avance500 spectrometer using a cryoprobe. Sequentialassignments of the peptide in the absence of m806Fab were establishedusing standard 2D TOCSY and NOESY as well as ¹⁵N-edited TOCSY and NOESYspectra. Interaction between the peptide and fAb806was examined bymonitoring ¹⁵N HSQC spectra of the peptide in the absence and presenceof fAb806. Spectral perturbation of ¹⁵N HSQC spectra of the peptide inthe presence of fAb806clearly indicates the peptide was able to bind tothe fAb806 under the presence solution conditions. Detailed conformationof the peptide in the complex form was not determined. Deviations fromrandom coil chemical shift values for the mAb806 peptide are shown inFIG. 93.

Biodistribution of chAb806 Tumor in Patients

To demonstrate the tumor specificity of mAb806 in vivo, a chimericversion (ch806) was engineered and produced under cGMP conditions(Panousis et al. (2005) Br. J. Cancer. 92, 1069-1077). A Phase Ifirst-in-man trial was conducted to evaluate the safety, biodistributionand immune response of ch806 in patients with 806 positive tumors, andthe results of safety, biodistribution and pharmacokinetics have beenreported previously (Scott et al. (2007) Proc. Natl. Acad. Sci. U.S.A.104, 4071-4076). To define the specificity of ch806 in tumor compared tonormal tissue (i.e., liver) in patients, the quantitative uptake ofch806 in tumor and liver was performed by calculation of % injected dose(ID) of ¹¹¹In-ch806 from whole body gamma camera images obtained overone week following injection of 5-7mCi (200-280MBq) ¹¹¹In-ch806. Liverand tumor dosimetry calculations were performed based on regions ofinterest in each individual patient. ¹¹¹In-ch806 infusion image dataset,corrected for background and attenuation, allowing calculation ofcumulated activity. Dosimetry calculation was performed to derive theconcentration of ¹¹¹In-ch806 in tumor and liver over a one week periodpost injection.

b. Sequencing

The variable heavy (VH) and variable light (VL) chains of mAb175 weresequenced, and their complementarity determining regions (CDRs)identified, as follows:

mAb175 VH chain: nucleic acid (SEQ ID NO:128) and amino acid (SEQ IDNO:129) sequences are shown in FIGS. 74A and 74B, respectively.Complementarity determining regions CDR1, CDR2, and CDR3 (SEQ IDNOS:130, 131, and 132, respectively) are indicated by underlining inFIG. 74B.

mAb175 VL chain: nucleic acid (SEQ ID NO:133) and amino acid (SEQ IDNO:134) sequences are shown in FIGS. 75A and 75B, respectively.Complementarity determining regions CDR1, CDR2, and CDR3 (SEQ ID NOS:135, 136, and 137, respectively) are indicated by underlining in FIG.75B.

The sequence data for mAb175 is based on both sequence and crystalstructure data, as the cell line is not clonal, and therefore multiplesequences have been obtained from the cell line. The sequences of mAb175set forth above have been confirmed by crystal structure, and differ bya single amino acid in each of the VL chain CDR1 and CDR2 from previoussequences based on standard sequence data alone. A different isotype ofmAb175 (an unusual IgG2a isotype) has also been obtained, based on thefinal sequence and crystal structure data.

mAb175 Specificity

Preliminary binding studies suggested that mAb175 displayed similarspecificity for EGFR as mAb806. In the CDR regions ofmAb806 (IgG2b) andmAb175 (IgG2a), the amino acid sequences are almost identical, with onlyone amino acid difference in each (FIG. 65; See Example 35, below). Allthese differences preserve the charge and size of the side-chains.Clearly these antibodies have arisen independently.

c. Experiments

A set of immunohistochemistry experiments were conducted to analyze thespecificity of mAb175 binding. mAb175 stains sections of A431 xenograftsthat overexpress the EGFR (FIG. 66A) and sections of U87MG.Δ2-7 gliomaxenografts that express the Δ2-7EGFR (FIG. 66A). In contrast, mAb175does not stain U87MG xenograft sections. The U87MG cell line onlyexpresses modest levels of the wild-type EGFR (FIG. 66A) and has nodetectable EGFR autocrine loop. Most importantly, mAb175 does not bindto normal human liver sections (FIG. 66B). Thus, mAb175 appears todemonstrate the same specificity as mAb806, i.e. it detectsover-expressed and truncated human EGFR, but not the wtEGFR expressed atmodest levels.

Identification of the mAb175 Epitope

Since mAb175 also binds the Δ2-7EGFR, in which amino acids 6-273 aredeleted, and EGFR₁₋₅₀₁, the mAb175 epitope must be contained withinresidues 274-501. When determining the epitope of mAb806, we expressed aseries of c-myc-tagged EGFR fragments fused to the carboxy terminus ofhuman GH, all terminating at amino acid 501 (Chao et al. (2004) J. Mol.Biol. 342, 539-550; Johns et al. (2004) J. Biol. Chem. 279,30375-30384).

The mAb175 also reacted with both the 274-501 and 282-501 EGFR fragmentsin Western blots, but did not detect fragments commencing at amino acid290 or 298 (FIG. 73). The presence of all GH-EGFR fusion proteins wasconfirmed using the c-myc antibody, 9E10 (FIG. 73). Therefore, acritical determinant of the mAb175 epitope is located near amino acid290. Finally, a 274-501 EGFR fragment with the mAb806 epitope deleted(Δ287-302) was also negative for mAb175 binding (FIG. 73), suggestingthat this region similarly determined most of the mAb175 binding.

A second approach was used to characterize the mAb175 epitope further.Fragments encompassing extracellular domains of the EGFR were expressedon the surface of yeast and tested for mAb175 binding by indirectimmunofluorescence using flow cytometry. The mAb175 recognized the yeastfragment 273-621, which corresponds to the extracellular domain of theΔ2-7 EGFR, but not to fragments 1-176, 1-294, 294-543, or 475-621 (FIG.67A and FIG. 67B). Thus, at least part of the mAb175 epitope must becontained within the region between amino acids 274-294, agreeing withimmunoblotting data using EGFR fragments. Since mAb175 binds to thedenatured fragment of the 273-621 (FIG. 67C), the epitope must be linearin nature (FIG. 73). It is clear that mAb806 and mAb175 recognize asimilar region and conformation of the EGFR.

Using surface plasmon resonance (BIAcore) the binding of mAb175 to theEGFR peptide (₂₈₇CGADSYEMEEDGVRKC₃₀₂; SEQ ID NO:138)) was investigated.The EGFR₂₈₇₋₃₀₂ was immobilized on the biosensor surface using amine,thiol-disulfide exchange or Pms-Ser coupling chemistries. The lattermethod immobilizes the peptide exclusively through the N-terminalcysteine (Wade et al. (2006) Anal. Biochem. 348, 315-317).

mAb175 bound the EGFR₂₈₇₋₃₀₇ in all orientations (Table 6). The affinityof mAb175 for EGFR₂₈₇₋₃₀₂ ranged from 35 nM for Pms-serine coupling to154 nM for amine coupling. In all cases the binding affinity of mAb175for EGFR₂₈₇₋₃₀₂ was lower than that obtained for mAb806 (Table 6). Wealso determined the affinity of mAb175 to two different extracellularfragments of the EGFR. mAb175 bound the 1-501 fragment with an affinitysimilar to that obtained using the peptide (16 nM versus 35 nM) (Table6). As expected, the affinity of mAb175 against the 1-621 full lengthextracellular domain, which can form the tethered conformation, was muchlower (188 nM). Although mAb806 and mAb175 have similar affinities forEGFR₂₈₇₋₃₀₂, mAb175 appears to display a higher affinity for theextra-cellular domain of the EGFR (Table 6). Clearly, the mAb175 epitopeis contained within the EGFR₂₈₇₋₃₀₂ and, like mAb806, the bindingaffinity to extra-cellular domain of the EGFR is dependent onconformation.

TABLE 6 BIAcore determination of antibody affinities for mAb806 andmAb175 binding to EGFR epitopes K_(D) for K_(D) for EGFR Fragment mAb175(nM) mAb806 (nM) 287-302 (Pms-Ser coupling) 35 16 287-302 (Thiolcoupling) 143 84 287-302 (Amine coupling) 154 85 1-501 (Unable to formtether) 16 34 1-621 (Can form tether) 188 389

The panel of mutants of the 273-621 EGFR fragment, expressed on thesurface of yeast (Chao et al. (2004) J. Mol. Biol. 342, 539-550; Johnset al. (2004) J. Biol. Chem. 279, 30375-30384) was used to characterizethe fine structure of the mAb175 epitope. mAb 175 and mAb806 displayed anear identical pattern of reactivity to the mutants (Table 7).Disruption of the 287-302 disulfide bond only had a moderate effect onthe epitope reactivity as the antibody bound to all mutants at C287 andto some but not all mutants at C302 (Table 7). Amino acids critical formAb175 binding include E293, G298, V299, R300 and C302 (Table 7). mAb175appeared moderately more sensitive to mutations V299 and D297 but mAb806also showed reduced binding to some mutations at these sites (Table 7).Again, the mAb175 epitope appears to be essentially the same as theepitope recognized by mAb806.

TABLE 7 Display of EGFR Epitope 287-302 mutations on yeast and thebinding scores for mAb806 and mAb175 EGFR Mutant mAb806 Binding mAb175Binding C287A + + C287G + + C287R + + C287S + + C287W + + C287Y + +G288A ++ ++ A289K ++ ++ D290A ++ ++ S291A ++ ++ Y292A ++ ++ E293A + +E293D + + E293G + + E293K — — M294A ++ ++ E295A ++ ++ E296A ++ ++ D297A++ + in contact D297Y + + G298A + + G298D — — G298S — — V299A ++ + incontact V299D — — V299K ++ + in contact R300A ++ ++ R300C + + R300P — —K301A ++ ++ K301E + + C302A — — C302F + + C302G — — C302R + + C302S — —C302Y + +Efficacy of mAb175 Against Tumor Xenografts Stimulated by Δ2-7EGFR or anEGFR Autocrine Loop

The in vivo anti-tumor activity of mAb806 and mAb175 against U87MG.Δ2-7glioma xenografts was examined. Xenografts were allowed to establish for6 days before antibody therapy (3 times a week for 2 weeks on daysindicated) commenced. At this time, the average tumor volume was 100 mm³(FIG. 68A). mAb175 treatment resulted in a reduction in overall tumorgrowth rate compared to treatment with vehicle or mAb806 and was highlysignificant at day 19 post-inoculation (P<0.0001 versus control andP<0.002 versus mAb806), when the control group was sacrificed forethical reasons. The average tumor volume at this time was 1530, 300 and100 mm³ for the vehicle, mAb806 and mAb175 treatment groups,respectively (FIG. 68A), confirming the antitumor activity of mAb175activity against xenografts expressing the Δ2-7 EGFR.

Even though U87MG cells express approximately 1×10⁵ EGFR per cell, mAb806 is not able to recognize any of the surface EGFR, and notsurprisingly, does not inhibit U87MG in vivo growth. Furthermore thesecells do not co-express any EGFR ligand. A study was conducted as towhether the EGFR epitope is transiently exposed, and hence able to berecognized by mAb806 and mAb175 in cells containing an EGFR autocrineloop. The prostate cell line DU145 expresses the wtEGFR at levelssimilar to that observed in U87MG cells, however unlike the U87MG cells,the DU145 cells contain an amplification of the TGF-α gene and thusexhibit an EGFR/TGF-α autocrine loop. Both mAb175 and 806 bind to DU145cells as determined by FACS analysis (FIG. 68B) and both are able toimmunoprecipitate a small proportion of the EGFR extracted from thesecells (FIG. 68C). Both techniques showed greater binding ofmAb175,however, when compared to mAb528, which binds to the L2 domain, mAb175and mAb806 only bind a subset of EGFR on the surface of these cells(FIG. 68B and FIG. 68C). Similar observations were seen with a secondprostate cell line (LnCap); (data not shown) and a colon line (LIM1215)both of which also contain EGFR autocrine loops (Sizeland, A. M. andBurgess, A. W. (1992) Mol Cell Biol. 3, 1235-1243; Sizeland, A. M. andBurgess, A. W. (1991) Mol Cell Biol. 11, 4005-4014). Clearly, mAb806 andmAb175 can recognize only a small proportion of the EGFR on cells in thepresence of an autocrine stimulation loop.

Since mAb175 and mAb806 bind more effectively to the EGFR expressed inDU145 cells than U87MG cells, a study was conducted to analyze theanti-tumor activity of these antibodies in DU145 xenografts grown innude mice. Xenografts were allowed to establish for 18 days beforetherapy commenced (3 times a week for 3 weeks on days indicated). Atthis time the average tumor volume was 90 mm³ (FIG. 68D). Both mAb175and mAb806 inhibited the growth of DU145 xenografts. The control groupwas sacrificed on day 67 and had a mean tumor volume of 1145 mm³compared with 605 and 815 mm³ for the mAb806 and mAb175 groupsrespectively (p<0.007 and 0.02 respectively) (FIG. 68D).

These results indicate that, while antibodies according to the presentinvention do not generally bind EGFR on cells expressing EGFR at normallevels (as discussed herein) they may bind EGFR on cells expressing EGFRat normal levels when such cells exhibit an EGFR autocrine loop, andfurthermore indicate that antibodies according to the present inventionare capable of inhibiting tumour growth in such cells.

3D-Structure of EGFR₂₈₇₋₃₀₇ in Contact with the Fab Fragments of mAb806and mAb175

In order to understand the molecular details of how mAb806 and mAb175could recognize EGFR in some, but not all conformations, the crystalstructures of Fab fragments for both antibodies were determined incomplex with the oxidized EGFR₂₈₇₋₃₀₂ epitope (at 2.0 and 1.59 Åresolution respectively, FIGS. 69A & 69B) and alone (at 2.3 Å and 2.8 Åresolution, respectively). In both cases, the free and complexed Fabstructures were essentially the same and the conformations of thepeptide and CDR loops of the antibodies were well defined (FIG. 69). Theepitope adopts a β-ribbon structure, with one edge of the ribbonpointing towards the Fab and V299 buried at the centre of theantigen-binding site (FIGS. 69C-E). Both ends of the epitope are exposedto solvent, consistent with these antibodies binding much longerpolypeptides.

Of the 20 antibody residues in contact with the epitope, there are onlytwo substitutions between mAb806 and mAb175 (FIG. 65). mAb175 contactresidues are: light-chain S30, S31, N32, Y49, H50, Y91, F94, W96 andheavy-chain D32, Y33, A34, Y51, S53, Y54, S55, N57, R59, A99, G100,R101; the mAb806 contact residues are the same, with sequencedifferences for the light-chain, N30 and heavy-chain, F33._(EGFR287-302) binds to the Fab through close contacts between peptideresidues 293-302, with most of the contacts being between residues 297and 302. The only hydrogen bonds between main chain atoms of EGFR₂₈₇₋₃₀₂and the Fab are for residues 300 and 302 (FIG. 69F). Recognition of theepitope sequence occurs through side-chain hydrogen bonds to residuesE293 (to H50 and R101 of the Fab), D297 (to Y51 and N57), R300 (to D32)and K301 (via water molecules to Y51 and W96). Hydrophobic contacts aremade at G298, V299 and C302.

The conformation of the epitope backbone between 293 and 302 wasessentially identical in the Fab806 and Fab175 crystals (rmsdeviation=0.4 Å, for Ca atoms in these residues). Although constrainedby the disulfide bond, the N-terminus of the peptide (287-292) does notmake significant contact in either antibody structure and conformationsin this region differ. However, this segment in the Fab806 complexappears rather disordered. More interestingly, the conformation of theEGFR₂₈₇₋₃₀₂ peptide in contact with the antibodies is quite closelyrelated to the EGFR₂₈₇₋₃₀₂ conformation observed in the backbone of thetethered or untethered EGFR structures (Li et al., 2005; Garrett et al.,2002). For EGFR₂₈₇₋₃₀₂ from the Fab175 complex, the rms deviations in Cαpositions are 0.66 and 0.75 Å, respectively (FIG. 69).

To gain further insight into the recognition of EGFR by mAb806 andmAb175, the conformation of ¹⁵N-labelled oxidized peptide EGFR₂₈₇₋₃₀₂was studied by NMR spectroscopy in solution, free and in the presence of806 Fab (see Materials and Methods). For the free peptide, resonanceswere assigned and compared to those for random coil. Essentially, thefree peptide adopted a random coil structure, not the beta ribbon asseen in the native EGFR (Garrett et al. (2002) Cell 20; 110, 763-773).

Upon addition of the Fab, resonance shifts were observed. However, dueto the weak signal arising from significant line broadening uponaddition of the Fab and successful crystallization of the complexes, thesolution structure of the Fab806-epitope complex was not pursuedfurther. Clearly though, when the peptide binds to the Fab fragment ofmAb806 (or mAb175) it appears that the Fab selects or induces theconformation of the peptide which matches that peptide in the nativereceptor.

In order to study why mAb806 and mAb175 recognize only someconformations of EGFR, the Fab fragment of mAb175 was docked onto anextra-cellular domain of EGFR (tethered and untethered monomers) bysuperimposing EGFR₂₈₇₋₃₀₇. For a Δ2-7-like fragment there were nosignificant steric clashes with the receptor. In the untethered formthere was substantially more accessible surface area of the Fab buried(920 Å² compared with 550^(Å2) in in the tethered form). Therefore, thisantigen may make additional contacts with non-CDR regions of theantibody, as has been indicated by yeast expression mutants (Chao et al.(2004) J. Mol. Biol. 342, 539-550). Conversely, docking the whole EGFRectodomain onto the Fab, there is substantial spatial overlap with thepart of the CR1 domain preceding the epitope (residues 187-286) andrunning through the centre of the Fab (FIGS. 69D and 69E). Hence, as theCR1 domain has essentially the same structure in tethered or untetheredconformations, mAb806 or mAb175 will be unable to bind to either form ofEGFR. Clearly, there must be a difference between the orientation of theepitope with respect to the CRI domain in either known conformations ofthe wtEGFR and the orientation that permits epitope binding. Inspectionof the CR1 domain indicated that the disulfide bond (271-283) precedingEGFR₂₈₇₋₃₀₂ constrains the polypeptide which blocks access to theepitope; disruption of this disulfide, even though it is not involved indirect binding to the antibodies, would be expected to allow partialunfolding of the CR1 domain so that mAb175 or mAb806 could gain accessto the epitope.

Breaking of the EGFR 271-283 Disulfide Bond Increases mAb806 Binding

Disulfide bonds in proteins provide increased structural rigidity but insome cell surface receptors, particularly those for cytokines and growthfactors, transient breaking of disulfide bonds and disulfide exchangecan control the receptor's function (Hogg, P. J. (2003) Trends inbiochemical sciences 28, 210-214). As this was one mechanism by whichmAb806 and mAb175 could gain access to their binding site, increasingthe accessibility of the epitope was attempted by mutating either orboth of the cysteine residues at positions 271 and 283 to alanineresidues (C271A/C283A). The vectors capable of expressing full lengthC271A-, C283A- or C271A/C283A-EGFR were transfected into the IL-3dependent Ba/F3 cell line. Stable Ba/F3 clones, which expressed theC271A- and C271A/C283A-EGFR mutant at levels equivalent to the wtEGFRwere selected (FIG. 70A. Ba/F3 cells expressing high levels of mutantC283A-EGFR were not observed. As previously described, the wtEGFR reactspoorly with mAb806; however, the mutant receptors reacted equallystrongly with mAb528, mAb806 and the anti-FLAG antibody, suggesting thatthe receptor is expressed at the cell surface, is folded correctly andthat the epitope for mAb806 is completely accessible in such cases. Toconfirm that mAb806 recognizes the C271A/C283A mutant more efficientlythan the wtEGFR, the ratio of mAb806 binding to the binding of mAb528was determined. Since both the wild-type and C271A/C283A EGFR wereN-terminally FLAG-tagged, the ratio of mAb806 and mAb528 binding to theM2 antibody was also determined. As reported previously, mAb806 onlyrecognized a small proportion of the total wtEGFR expressed on thesurface of Ba/F3 cells (the mAb806/528 binding ratio is 0.08) (Table 8).In contrast, mAb806 recognized virtually all of the C271A/C283A mutantEGFR expressed on the cell surface (an mAb806/528 binding ratio of 1.01)(FIG. 70A and Table 8).

TABLE 8 mAb806 reactivity with cells expressing the wild-type orC271A/C283A EGFR Ratios of antibody binding Cell Line mAb 528/M2mAb806/M2 mAb806/mAb 528 wtEGFR- 1.37 0.11 0.08 FLAG wt-EGFR — — 0.07C271/283* 1.08 ± 0.10 1.09 ± 0.38 1.01 ± 0.13 *Average for fourindependent clones

Mutation of the two cysteines did not compromise EGF binding or receptorfunction. BaF3 cells expressing the C271A/C283A EGFR mutant proliferatein the presence of EGF (FIG. 70B). A left-shift in the dose responsecurve for EGF in cells expressing the C271A/C283A mutations wasreproducibly observed, suggesting either higher affinity for the ligand,or enhanced signaling potential for the mutant receptor. Westernblotting analysis confirmed that the C271A/C283A mutant is expressed atsimilar levels to the wtEGFR and is tyrosine phosphorylated in responseto EGF stimulation (FIG. 70C). Consistent with previous studies in othercell lines, mAb806 has no effect on the in vitro EGF-inducedproliferation of Ba/F3 cells expressing the wtEGFR, while the ligandblocking mAb528 completely inhibits the EGF-induced proliferation ofthese cells (FIG. 70D, left panel). In contrast, mAb806 totally ablatedthe EGF-induced proliferation in BaF3 cells expressing the C271A/C283Amutant (FIG. 70D, right panel). When the 271-283 cysteine loop isdisrupted, not only does mAb806 bind more effectively, but once bound,mAb806 prevents ligand induced proliferation.

TABLE 9 Data Collection and Refinement Statistics 806 (native) 806(peptide) 175 (native) 175 (peptide) Data Collection Space Group P2₁2₁2P2₁ P2₁2₁2₁ P2₁2₁2 Cell Dimensions (Å) a 140.37 35.92 36.37 83.17 b74.62 83.16 94.80 69.26 c 83.87 72.21 β = 92.43 108.90 71.47 Sourcein-house BNL X29 in-house in-house Wavelength (Å) 1.542 1.1 1.542 1.542Resolution Range (Å) 29.7-2.2    50-2.0 50-2.8 14.18-1.59  (2.27-2.20)(2.07-2.0) (2.87-2.8)   (1.65-1.59) R_(merge) (%)  6.4 (26.7)  6.6(28.2)  8.6 (30.0) I/σI 12.2 (3.2)    22 (3.15) 10.2 (2.2)  Completeness(%) 98.3 (91.3) 96.6 (79.2) 98.4 (90.5) 78.8 (11.8) 98.1 at 1.89 Å TotalReflections 156497 98374 205401 Unique Reflections 44905 27692 917143879 Refinement Resolution range (Å)  20-2.3 72.17-2.00 50-2.614.18-1.6  Reflections 37397 26284 9171 41611 R_(cryst) 0.225 0.2260.210 0.203 R_(free) 0.289 0.279 0.305 0.257 Protein Atoms 6580 32943276 3390 Solvent Atoms 208 199 46 247 r.m.s.d bond length (Å) 0.0220.007 0.015 0.014 r.m.s.d bond length (°) 1.70 1.12 1.77 1.48 AverageB-factor (Å²) 40.3 33.6 37.5 20.7 Overall anisotrpic −1.52 2.42 0.201.13 B-factors (Å²) B11

Discussion

Structural studies with the EGFR₂₈₇₋₃₀₂ epitope show that both mAb806and mAb175 recognized the same 3D-structural motif in the wtEGFRstructures, indicating that this backbone conformation also occurs inand is exposed in the Δ2-7EGFR. Critically, however, the orientation ofthe epitope in these structures would prevent antibody access to therelevant amino acids. This is consistent with the experimentalobservation that mAb806 does not bind wtEGFR expressed on the cellsurface at physiological levels.

The results with the EGFR_(C271A/C283A) mutant indicate that the CR1domain can open up to allow mAb806 and mAb175 to bind stoichiometricallyto this mutant receptor. This mutant receptor can still adopt a nativeconformation as it is fully responsive to EGF stimulation but, unlikethe wtEGFR, is fully inhibited by mAb806. If a misfolded form of theEGFR with this disulfide bond broken were to exist on the surface ofcancer cells, the data clearly shows it would be capable of initiatingcell signaling and should be inhibited by either mAb806 or mAb175.

Another explanation of the data is that during ligand activation thestructural rearrangement of the receptor could induce local unfolding inthe vicinity of the epitope, allowing the receptor to adopt aconformation which permits binding. In crystal structures, the epitopelies near the physical centre of the EGFR ectodomain and access to theepitope is blocked by both the folded CR1 domain and the quaternarystructure of the EGFR ectodomain. In the tethered and the untetheredconformations, the integrity of the CR1 domain is stabilized byadditional interactions with either the L1:ligand:L2 domains(untethered) or the L2:CR2 domains (tethered). However, the epitoperegion has some of the highest thermal parameters found in theectodomain: the mAb806/175 epitope is structurally labile. Duringreceptor activation, when the receptor undergoes a transition betweenthe tethered and untethered conformations, mAb806 and mAb175 can accessthe epitope. Thus at the molecular level, these mechanisms couldcontribute to the negligible binding of mAb806 and mAb175 to normalcells and the substantially higher levels of binding to tumor cellswhich have overexpressed and/or activated EGFR.

EXAMPLE 24 Monoclonal Antibodies 124 and 1133

As discussed in Example 1 above, mAb124 and mAb1133 were generated atthe same time as mAb806 and found to display similar properties, inparticular specificity for the over-expressed wild-type EGFR, to theunique properties of mAb806 discussed herein.

Initial screens were conducted in New York (Jungbluth et al. (2003) AMonoclonal Antibody Recognizing Human Cancers withAmplification/Over-Expression of the Human Epidermal Growth FactorReceptor PNAS. 100, 639-644. ELISA competition assessments and Biacoreanalyses were conducted to determine whether mAb124 and/or mAb1133recognize an epitope identical to mAb806 or an alternative EGFRdeterminant.

FACS Analysis

Antibody binding to U87MG.Δ2-7, A431 and HN5 cells was assessed by FACS.All antibodies displayed a similar specificity as that of mAb806 withstrong binding to the de2-7 EGFR and low binding to over-expressedwild-type EGFR.

Competition ELISA

A series of competition ELISAs were conducted to determine whether the124 and 1133 antibodies competed with the mAb806 epitope. Briefly, thedenatured soluble domain of the EGFR (sEGFR) was coated on to ELISAplates. The unlabeled 124 or 1133 antibodies were then added across theplate in increasing concentrations. Following washing, biotinylatedmAb806 was added to each well to determine if it could still bind thesEGFR. Detection of bound mAb806 was achieved usingstreptavidin-conjugated HRP. If an antibody binds the same (oroverlapping) epitope as mAb806 then mAb806 binding is not expected.

Results are summarized in Table 10. A concentration dependant inhibitorybinding effect was observed for mAb124 and mAb1133: mAb806 bindingincreased as concentration of unlabeled antibody was decreased,suggesting that the 124 and 1133 antibodies recognize an epitopeidentical to mAb806 or one in close proximity.

TABLE 10 Summary mAb124 and mAb1133 Competition ELISA binding to sEGFRUnlabeled Blocking Antibody Binding of biotin-labeled 806 124 None 1133None 806 (control for inhibition) None Irrelevant IgG2b ++++

FACS Analysis: Cell Binding Competition

U87MG.Δ2-7 cells were pre-incubated with unlabeled antibody 124, 1133.Positive control 806 and isotype control were included in the assay.Cells were washed, then stained with Alexa488-conjugated mAb806 and thelevel of 806 binding was determined by FACS.

Results are summarized in Table 11. The 124 and 1133 antibodies blockedmAb806 binding to the cell surface indicating recognition of an epitopeidentical to mAb806 or one in close proximity.

TABLE 11 FACS Analysis: U87MG.Δ2-7 Cell Binding Competition UnlabeledInhibition of Blocking Antibody Alexa488-labeled 806 124 +++ 1133 +++806 ++++ IgG2b control noneBIAcore Analysis: Binding to the mAb806 Peptide Epitope

The EGFR amino acid sequence ₂₈₇CGADSYEMEEDGVRKC₃₀₂ (SEQ ID NO:14)containing the mAb806 epitope was synthesized as a peptide andimmobilized onto the biosensor chip. Binding of antibodies 124, 1133 and806 (200 nM) to this peptide was measured. Maximal binding resonanceunits (RU) obtained are summarized in Table 12. The 124, 1133 showedclear binding to the peptide confirming recognition of the 806 peptideepitope.

TABLE 12 BIAcore Analysis: Maximal binding to the mAb806 peptide epitopeBinding to mAb806 Antibody peptide (RU) 806 1100 124 1000 1133 800

Discussion

As shown in this Example, mAb124 and mAb1133 bind to the EGFR peptiderecognized by mAb806 and block binding of mAb806 to the extracellulardomain of EGFR and cells expressing the de2-7 EGFR. Thus, these threeantibodies recognize the same determinant on EGFR.

EXAMPLE 25 Monoclonal Antibody 585

As discussed in Example 1 above, mAb585 was generated at the same timeas mAb806 and found to display similar properties, in particularspecificity for the over-expressed wild-type EGFR, to the uniqueproperties of mAb806 discussed herein.

Initial screens were conducted in New York (Jungbluth et al. (2003) AMonoclonal Antibody Recognizing Human Cancers withAmplification/Over-Expression of the Human Epidermal Growth FactorReceptor PNAS. 100, 639-644.

Biacore analysis was conducted to determine if mAb585 recognizes anepitope identical to mAb806 or an alternative EGFR determinant.

BIAcore Analysis: Binding to the mAb806 Peptide Epitope

The EGFR amino acid sequence ₂₈₇CGADSYEMEEDGVRKC₃₀₂ (SEQ ID NO:14)containing the mAb806 epitope was synthesized as a peptide andimmobilized onto the biosensor chip. Binding of antibody 585 and 806(200 nM) to this peptide was measured. Maximal binding resonance units(RU) obtained are summarized in Table 13. The 585 antibody showed clearbinding to the peptide confirming recognition of the 806 peptideepitope.

TABLE 13 BIAcore Analysis: Maximal binding to the mAb806 peptide epitopeBinding to mAb806 Antibody peptide (RU) 806 1100 585 450

Discussion

As shown in this Example, mAb585 binds to the EGFR peptide recognized bymAb806. Thus, these antibodies recognize the same determinant on EGFR.

EXAMPLE 26 Clinical Testing of ch806

A clinical study was designed to examine the in-vivo specificity ofch806 in a tumor targeting/biodistribution/pharmacokinetic analysis inpatients with diverse tumor types.

1. Materials and Methods Trial Design

This first-in-man trial was an open label, dose escalation Phase Istudy. The primary objective was to evaluate the safety of a singleinfusion of ch806 in patients with advanced tumors expressing the 806antigen. The secondary study objectives were to determine thebiodistribution, pharmacokinetics and tumor uptake of ¹¹¹In-ch806;determine the patient's immune response to ch806; and to assess earlyevidence of clinical activity of ch806. A single dose was chosen forthis study in order to optimally assess the in-vivo specificity of ch806for EGFR expressed on tumor. The protocol was approved by the HumanResearch and Ethics Committee of the Austin Hospital prior to studycommencement. The trial was performed under the Australian TherapeuticGoods Administration Clinical Trials Exemption (CTX) scheme. Allpatients gave written informed consent.

Eligibility criteria included: advanced or metastatic tumors positivefor 806 antigen expression based on chromogenic in-situ hybridisation orimmunohistochemistry of archived tumor samples (tumors were defined as806 positive if immunohistochemical assessment of archived tumoursamples showed any cells positive for 806 expression, see below);histological or cytologically proven malignancy; measurable disease onCT scan with at least one lesion ≧2 cm; expected survival of at least 3months; Karnofsky performance scale (KPS)≧70; adequate hematologic,hepatic and renal function; age>18 yrs; and able to give informedconsent. Exclusion criteria included: active central nervous systemmetastases (unless adequately treated and stable); chemotherapy,immunotherapy, biologic therapy, or radiation therapy within four weeksprior to study entry; prior antibody exposure [unless no evidence ofhuman anti-chimeric antibodies (HACA)]; failure to fully recover fromeffects of prior cancer therapy; concurrent use of systemiccorticosteroids or immunosuppressive agents; uncontrolled infection orother serious disease; pregnancy or lactation; women of childbearingpotential not using medically acceptable means of contraception.

Patients received a single infusion of ch806 trace labelled withIndium-111 (¹¹¹In, 200-280 MBq; 5-7 mCi) by intravenous infusion innormal saline/5% human serum albumin over 60 minutes. The planned doseescalation meant patients were enrolled into one of four dose levels: 5,10, 20 and 40 mg/m². These doses were chosen to allow assessment of thespecificity of ch806 to EGFR expressed on tumor, and to determine if anynormal tissue compartment binds ch806 (and affects pharmacokinetics orbiodistribution) in-vivo. Biodistribution, pharmacokinetics, and immuneresponse were evaluated in all patients.

Whole body gamma camera imaging for assessment of biodistribution andtumour uptake was performed on Day 0, Day 1, Day 2 or 3, Day 4 or 5, andDay 6 or 7 following ¹¹¹In-ch806 infusion. Blood samples forpharmacokinetics were obtained at these time-points, and additionally onDay 14 (±2 days) and Day 21 (±2 days). Blood samples for assessment ofHACA levels were obtained at baseline, and weekly until Day 30. Toxicityassessment was performed at each study visit. Physical examination androutine hematology and biochemistry were performed weekly until end ofstudy (Day 30). Restaging was performed on Day 30.

Dose Escalation Criteria

The first patient at each dose level was observed for four weeks priorto enrollment of any additional patients. If no dose limiting toxicity(DLT) was observed in any of the first 2 patients within 4 weeks of theinfusion of ch8063, 4 patients were then to be entered on the nexthighest dosage tier. If one patient in any cohort of 2 patientsexperienced a DLT within 4 weeks from the first dose, an additional 4patients (maximum of 6) were entered at that dosage level. If no morethan one patient out of 6 in any dose level experienced≧Grade 3toxicity, subsequent patients were entered at the next dose level.

DLT was defined as Grade 3 non-haematological toxicity, or Grade 4haematological toxicity as defined by the NCI Common TerminologyCriteria for Adverse Events (CTCAE v3.0). Maximum tolerated dose (MTD)was defined as the ch806 dose below that where 2 or more patients out of6 experienced DLT.

Radiolabeling of Ch806

Clinical grade ch806 was produced in the Biological Production Facilityof the Ludwig Institute for Cancer Research, Melbourne, Australia. Theantibody ch806 was labelled with ¹¹¹In (MDS Nordion, Kanata, Canada) viathe bi-functional metal ion chelate CHX-A″-DTPA according to methodsdescribed previously (Scott et al. (2000) Cancer Res 60, 3254-3261;Scott et al. (2001) J. Clin. Oncol. 19(19), 3976-3987).

Gamma Camera Imaging

Whole body images of ¹¹¹In-ch806 biodistribution were obtained in allpatients on Day 0 after infusion of ¹¹¹In-ch806, and on at least 3further occasions up to Day 7 following infusion. Single photon emissioncomputed tomography (SPECT) images of a region of the body with knowntumor were also obtained on at least one occasion during this period.All gamma camera images were acquired on a dual-headed gamma camera(Picker International, Cleveland, Ohio).

Pharmacokinetics

Blood for pharmacokinetic analysis was collected on Day 0-pre¹¹¹In-ch806 infusion; then at 5 minutes, 60 minutes, 2 h and 4h post¹¹In-ch806 infusion, Day 1, Day 2 or 3, Day 4 or 5, and Day 6 or 7.Further blood for pharmacokinetics of ch806 protein was also obtained onDay 14 (±2 days) and Day 21 (±2 days) and Day 30 (±2 days).

Serum samples were aliquoted in duplicate and counted in a gammascintillation counter (Packard Instruments, Melbourne, Australia), alongwith appropriate ¹¹¹In standards. The results of the serum wereexpressed as % injected dose per litre (% ID/L). Measurement of patientserum ch806 protein levels following each infusion was performed using avalidated protocol for the immunochemical measurement of ch806 proteinin human serum⁴⁰. The limit of quantitation for ch806 in serum sampleswas 70 ng/mL. All samples were assayed in triplicate and were diluted bya factor of at least 1:2. Measured serum levels of ch806 were expressedas μg/mL.

Pharmacokinetic calculations were performed on serum ¹¹¹In-ch806measurements following the infusion, and ELISA determined patient serach806 protein levels, using a curve fitting program (WinNonlin Pro Node5.0.1, Pharsight Co., Mountain View, Calif.). Estimates were determinedfor the following parameters: T½α and T½↑ (half lives of the initial andterminal phases of disposition); V1, volume of central compartment;C_(max) (maximum serum concentration); AUC (area under the serumconcentration curve extrapolated to infinite time); and CL (total serumclearance).

Whole Body Clearance and Tumor and Organ Dosimetry of ¹¹¹In-ch806

Whole body and normal organ (liver, lungs, kidney and spleen) dosimetrycalculations were performed based on regions of interest in eachindividual patient ¹¹¹In-ch806 infusion image dataset, allowingcalculation of cumulated activity and analysis using OLINDA for finaldosimetry results (Stabin et al. (2005) J. Nucl. Med. 46(6), 1023-1027).Regions of interest were also defined for suitable tumors at each timepoint on ¹¹¹In-ch806 image datasets, corrected for background andattenuation, and dosimetry calculation was performed to derive theconcentration of ¹¹¹In-ch806 in tumor/gm (Scott et al. (2005) Clin.Cancer Res. 11(13), 4810-4817). This was converted to μg ch806/gm tumortissue based on the injected mg ch806 protein dose.

HACA Analysis

Blood samples for HACA assessment were taken prior to ch806 infusion,then weekly until 30 days after ch806 infusion. Samples were analysed byELISA, and by surface plasmon resonance technology using a BIAcore2000instrument, as described previously (Scott et al., 2005; Liu et al.(2003) Hybrid Hybridomics 22(4), 219-28; Ritter et al. (2001) CancerRes. 61(18), 685-6859).

Immunohistochemistry Method

Formalin-fixed paraffin embedded tumor tissue from each patient on thetrial was immunostained as follows: Briefly, 4 μm sections of paraffinembedded tissue were mounted onto SuperFrost® Plus slides(Menzel-Glaser, Germany), de-paraffinized and rehydrated prior tomicrowave antigen retrieval in Target Retrieval Solution, pH 6.0 (10min; Dako, Glostrup, Denmark). Sections were then treated with 3% H2O2for 10 min, to eliminate endogenous peroxidase and incubated at roomtemperature for 60 min with m806 antibody (4 μg/ml) or with appropriateconcentration of isotype-matched negative control antibody (IgG2b;Chemicon, Temecula, Calif.). Antibody binding was detected using thePowerVision Kit (ImmunoVision Technologies, Brisbane, Calif.). To allowvisualization of the immunostaining, sections were incubated with thechromogen 3-amino-9-ethylcarbazole (0.4%, Sigma Chemical Co. MO, USA)for 10 min and counterstained with Mayer's haematoxylin. Negativecontrols for the immunostaining procedure were prepared by omission ofthe primary antibody. Results were expressed as a percentage of positivetumor cell staining

Chromogenic in Situ Hybridization Method

Formalin fixed paraffin embedded tumor tissue from each patient on thetrial was sectioned and mounted on SuperFrost® Plus slides,de-paraffinized and rehydrated prior to pre-treatment with theSpotLight® Tissue Pre-treatment Kit (Zymed Laboratories Inc. South SanFrancisco, Calif.). Sections were then covered with the SpotLight® EGFRDNA probe, denatured at 95° C. for 10 min and incubated overnight at 37°C. Following hybridization, slides were washed in 0.5×SSC. Detection ofthe probe was carried out using the SpotLight® CISH™ Polymer DetectionKit. Sections that showed clusters of signals or ≧5 individual signalsin >25% of cancer cells were considered to have an amplification of theEGFR gene that correlated with m806 reactivity.

2. Results Patients

Eight patients (1 female and 7 male; mean age of 61 years (range 44-75)]completed the trial (Table 14). Primary tumor sites, prior therapyhistory, and sites of disease at study entry are also shown in Table 14.All 8 patients had 806 antigen positivity in archived tumors (Table 14).

All patients fulfilled inclusion criteria and, except for Patient 8 (whohad a primary brain tumor), all had metastatic disease at study entry.Sites of disease classified as target lesions included: lung (5patients), brain (1 patient), lymph nodes (1 patient), supraglottis (1patient). Other sites of metastatic disease (non-target lesions)included a supra-renal mass, bone and lymph nodes (Table 14). The medianKarnofsky performance status was 90 (range 80-100).

TABLE 14 Patient Characteristics Dose IHC of Disease Tumor Pt. Level AgeKPS Site of Primary positive Prior Sites at response to No. (mg/m²)(yrs) Sex (%) Tumour cells (%) Therapies Study Entry ch806 1 5 71 M 10NSCLC 50-75 RT Lung, PD Adrenal 8 5 44 M 90 Anaplastic  >75* Surgery,Brain SD astrocytoma RT, CT 2 10 49 F 80 SCC Anus <10 Chemo, LN, Lung,SD RT Bone 3 10 75 M 90 NSCLC 50-75 Surgery Lung SD RT 4 20 52 M 100Colon  <10† Surgery, Lung, LN PD CT 5 20 65 M 80 Mesothelioma >75 RT, CTLung SD 6 40 59 M 80 SCC vocal cord >75 Surgery, Soft Tissue SD RT, CT 740 71 M 90 SCC skin 50-75 Surgery, Lung, LN PD CT Abbreviations: F =female; M = male; NSCLC = non small cell lung carcinoma; SCC = squamouscell carcinoma; RT = radiotherapy; CT = chemotherapy; LN = lymph nodes;PD = progressive disease; SD = stable disease *positive for de2-7 EGFRexpression †positive for EGFR gene amplification

Adverse Events and HACA

Adverse events related to ch806 are listed in Tables 17 and 18. Noinfusion related adverse events were observed. There was no DLT, andhence MTD was not reached. The principle toxicities that in theinvestigator's opinion were possibly attributable to ch806 were:transient pruritis, mild nausea, fatigue/lethargy, and possible effectson serum ALP and GGT levels. A CTC grade 2 elevation in GGT level inPatient 5 was observed, however this was on a background of a baselinegrade 1 elevation, and was transient in nature. Three serious adverseevents (SAEs) were reported but none were attributed to ch806. Overall,ch806 was safe and well tolerated at all dose levels with generallypredictable and manageable minor toxicities being observed. Further doseescalation was not performed due to the limited amount of cGMP ch806available for the trial.

A positive immune response to ch806 (with concordance of both ELISA andBIAcore methodologies) was observed in only one of the eight patients(Patient 1).

TABLE 15 Occurrence of Adverse Events Related to ch806 Dose Level(mg/m²)* Total Number of Episodes Adverse Event 5 10 20 40 of Each EventDizziness 0 0 0 1 1 Fatigue 0 0 1 0 1 Lethargy 0 0 0 1 1 Appetite 0 0 01 1 suppressed Nausea 0 1 0 1 2 Pruritis 1 0 0 0 1 ALP - elevated 0 0 10 1 GGT - elevated 0 0 1 0 1 Total 1 1 3 4 9 *Numbers represent numberof episodes of any event at each dose level

TABLE 16 Distribution of Study Agent Related Adverse Events Maximum CTCGrade Toxicity* Dose Level 4 = Life- (mg/m²) 1 = Mild 2 = Moderate 3 =Severe threatening 5 1 0 0 0 10 1 0 0 0 20 2 1 0 0 40 4 0 0 0 Overall 81 0 0 *Number of patientsRadiolabeling of ch806

There were a total of 8 infusions of ¹¹¹In-ch806 administered during thetrial. The mean (±SD) radiochemical purity and immunoreactivity of¹¹¹In-ch806 was measured to be 99.3±0.1% and 77.4±7.0% respectively.

Biodistribution of ch806

The initial pattern of ¹¹¹In-ch806 biodistribution in patients at alldose levels was consistent with blood pool activity, which clearedgradually with time. Over the one week period post injection the uptakeof ¹¹¹In-ch806 in liver and spleen was consistent with the normalclearance of ¹¹¹In-chelate metabolites through the reticuloendothelialsystem. Specific localization of ¹¹¹In-ch806 was observed in targetlesions (≧2 cm) of all patients at all dose levels (FIG. 94), includingtarget lesions located in the lungs (Patients 1, 3, 4, 5, and 7), theabdomen (Patients 1 and 2), and the supraglottic region in the rightside of the neck (Patient 6). High uptake of ¹¹¹In-ch806 in a braintumor (Patient 8) was also demonstrated (FIG. 95). Importantly, uptakeof ¹¹¹In-ch806 in tumor was not dependent on a the level of 806 antigenexpression. For example, Patient 4 demonstrated high uptake by both lungtarget lesions, despite <10% positivity by IHC for 806 reactivity inarchived tumor (FIG. 96). This degree of uptake of ¹¹¹In-ch806 in targetlesions in Patient 4 was comparable to that seen in Patient 3, where50-75% of tumor cells were positive for 806 antigen staining on archivedsample immunohistochemistry (FIG. 96).

Pharmacokinetics

Individual patient pharmacokinetic parameters T½α and T½β, V1, C_(max),AUC and CL for the single infusion of ¹¹¹In-ch806 are shown in Table 17.The Kruskal-Wallis rank sum test was applied to the alpha and beta halflives, V1 and clearance. No significant difference between dose levelswas observed (P>0.05).

The pharmacokinetic curve fit to the pooled population ELISA data isshown in FIG. 97. The mean±SD pharmacokinetic parameters were T½α29.16±21.12 hrs, T½β 172.40±90.85 hrs, V1 2984.59±91.91 ml, and CL19.44±4.05 ml/hr. Measured peak and trough ch806 serum concentrations(C_(max) and C_(min)) data are presented in Table 18 for each patient.As expected, linear relationships were observed for C_(max) and C_(min)with each dose level. The mean±SD values determined for the ch806 ELISApharmacokinetic data were in good agreement with the values obtained forthe ¹¹¹In-ch806 pharmacokinetic data (Table 17).

TABLE 17 Mean ± SD Pharmacokinetic Parameter Estimates for¹¹¹In-CHX-A″-DTPA-ch806 in each Dose Level and across all Dose Levels. T½ α T ½ β V1 CL AUC Dose Level (hr) (hr) (mL) (mL/hr) (hr*mg/mL) (mg/m²)Mean SD Mean SD Mean SD Mean SD Mean SD 5 10.91 3.4 183.9 110.2 2963.06493.23 21.97 16.59 541.17 371.75 10 11.75 4.4 124.5 9.25 3060.29 721.7028.58 8.60 566.79 26.39 20 9.34 8.3 125.3 73.66 2902.06 1064.77 30.9821.65 1438.12 957.18 40 8.95 3.2 133.9 10.79 4742.42 169.10 37.99 6.472269.04 381.68 ALL 10.24 1.32 141.90 28.30 3416.96 886.04 29.88 6.61

TABLE 18 Cmax and Cmin Serum ch806 Levels Determined by ELISA Analysis.DOSE LEVEL C_(max)* C_(min)* PT. NO. (MG/M²) (μG/ML) (μG/ML) 1 5  1.38 ±0.02  0.10 ± 0.05† 8 5  1.52 ± 0.17 0.96 ± 0.08 2 10  5.92 ± 0.11 1.50 ±0.01 3 10  6.27 ± 0.45 1.83 ± 0.20 4 20 12.25 ± 0.66 4.05 ± 0.05 5 2011.22 ± 0.77 1.58 ± 0.04 6 40 27.76 ± 2.10 6.90 ± 0.38 7 40 32.32 ± 0.846.80 ± 0.13 *C_(max) = 60 min post injection.; C_(min) = Day 7 †Day 8serum level

Dosimetry of ¹¹¹In-ch806

Whole body clearance was similar in all patients across all dose levels,with a T_(1/2)biologic (mean±SD) of 948.6±378.6 hrs. Due to therelatively short physical half-life, calculation of biological halftimewas extremely sensitive to small changes in effective halftime. Therewas no statistical significant difference in whole body clearancebetween dose levels [Kruskal-Wallis rank sum test: P-value=0.54] (FIG.98).

The clearance of ¹¹¹In-ch806 from normal organs (liver, lungs, kidneyand spleen) showed no difference between dose levels, and the meanT_(1/2)effective was calculated to be 78.3, 48.6, 69.7 and 66.2 hrsrespectively. There was no statistically significant difference inclearance between these normal organs. In particular, liver clearanceshowed no difference between dose levels (FIG. 98), indicating nosaturable antigen compartment in the liver for ch806.

Tumor dosimetry analysis was completed for 6 patients. Patients 1 and 2had target lesions close to the cardiac blood pool, or motion duringsome image acquisitions, which prevented accurate analysis. The measuredpeak uptake of ¹¹¹In-ch806 occurred 5-7 days post infusion, and rangedfrom 5.2-13.7×10⁻³% injected dose/gm tumor tissue.

Assessment of Clinical Activity

At the completion of this one month study period 5 patients were foundto have stable disease, and 3 patients progressive disease (Table 14).Interestingly, one patient (Patient 7, 40 mg/m² dose level) had clinicalevidence of transient shrinkage of a palpable auricular lymph node(proven to be metastatic SCC on fine needle aspiration) during the studyperiod, which suggests possible biologic activity of ch806. However,this patient had confirmed progressive disease by RECIST at studycompletion.

Additional Data

Eight patients [1 female and 7 male; mean age of 61 years (range 44-75)]completed this phase 1 trial as reported (Scott et al. (2007) Proc.Natl. Acad. Sci. U.S.A. 104, 4071-4076). All patients fulfilledinclusion criteria and, except for Patient 8 (who had a primary braintumor), all had metastatic disease at study entry. Ab uptake by thetumor was seen in all patients, and ¹¹¹In-ch806, the chimerized versionof mAb806, demonstrated prompt and high level uptake in tumor (FIG. 71).The clearance of ¹¹¹In-ch806 from normal organs (liver, lungs, kidneyand spleen) showed no difference between dose levels (Scott et al.,2007). In particular, liver clearance showed no difference between doselevels, indicating no saturable antigen compartment in the liver forch806. Total liver uptake was a maximum of 14.45±2.43% ID immediatelypost infusion, and declined to 8.45±1.63% ID by 72 hours, and 3.18±0.87%ID by one week post infusion. This is in marked contrast to the uptakeof antibodies to wtEGFR (e.g. 225), which have been shown to reach over30% ID in liver (for a 40 mg dose) for over 3 days post infusion (Divgiet al. (1991) J. Natl. Cancer Inst. 83, 97-104). The measured peak tumoruptake of ¹¹¹In-ch806 occurred 5-7 days post infusion. Calculation ofquantitative tumor uptake in Patients 1 and 3 could not be accuratelyperformed due to proximity of target lesion to cardiac blood pool andpatient movement. Peak ch806 uptake in tumor ranged from 5.21 to13.73×10⁻³% ID/gm tumor tissue. Calculation of actual ch806concentration in tumor showed peak values of (mean±SD) 0.85±0 μg/gm (5mg/^(m2)), 0.92±0 μg/gm (10 mg/^(m2)), 3.80±1.10 μg/gm (20 mg/m²), and7.05±1.40 μg/gm (40 mg/m²).

Discussion

As set forth in this Example, this study represents the first reporteddemonstration of the biodistribution and tumor targeting of a chimericantibody against an epitope only exposed on overexpressed, mutant orligand activated forms of the EGFR. Ch806 showed excellent targeting oftumor sites in all patients, no evidence of normal tissue uptake, and nosignificant toxicity. These in vitro and in vivo characteristics ofch806 distinguish it from all other antibodies targeting EGFR.

At doses up to 40 mg/m², ch806 was well tolerated, no DLT was observedand MTD was not reached. The principle toxicities that were possiblyattributable to ch806 were transient pruritis, mild nausea,fatigue/lethargy, and possible effects on serum ALP and GGT levels. Theadvanced nature of these patient's malignancies meant their diseasecould also have been contributing factors to these adverse events. Ofthe adverse events that were possibly related to study drug, all weremild, many were self-limiting, and none required any active treatment.Importantly, no skin rash or gastrointestinal tract disturbances wereobserved in any patient, even at the highest dose level. The excellenttolerability of ch806 in this single-dose study justifies the next stepof testing in repetitive dose trials.

The biodistribution of ch806 in all patients showed gradual clearance ofblood pool activity, and no definite normal tissue uptake of¹¹¹In-ch806. Excellent tumor uptake of ch806 was also evident in allpatients, including lung, lymph node, and adrenal metastases, and inmesothelioma and glioma. This was observed at all dose levels including5 mg/m² (the lowest dose studied), which is one tenth to one twentiethof the dose required to visualise uptake in tumor by other antibodies towtEGFR³³. This difference in uptake of ch806 compared to antibodies towtEGFR can be attributed to their substantial normal tissue (liver andskin) uptake due to wtEGFR acting as an antigen sink³³. In addition, thelocalization of ¹¹¹In-ch806 was high even in patients with lowexpression of 806 assessed by immunohistochemistry of archived tumorsamples (FIG. 96). The uptake of ¹¹¹In-ch806 in glioma was particularlyimpressive (FIG. 97), and comparable to any published data on antibodytargeting of brain tumor following systemic or even locoregionalinfusion. This data supports the unique selectivity of ch806 to EGFRexpressed by a broad range of tumors, and confirms the lack of normaltissue uptake of this antibody in human.

Pharmacokinetic analyses showed that ch806 has a terminal half-life ofmore than a week, and no dose dependence of ¹¹¹In-ch806 serum clearance.Linear relationships also were observed for AUC, Cmax and Cmin, withdose levels above 10 mg/m² achieving trough serum concentrations above 1μg/mL. The V1, Cl, T½α and T½β values were consistent between doselevels, and in keeping with typical IgG1 human antibodies (Scott et al.,2005; Steffens et al. (1997) J. Clin. Oncol 15, 1529-1537; Scott et al.(2001) J. Clin. Oncol. 19(19), 3976-3987). The clearance of ch806 wasalso determined to be slower when ELISA ch806 calculations were comparedto ¹¹¹In-ch806 measurements. While this difference may be explained bythe small number of patients studied, the longer sampling time pointsfor the ch806 ELISA would support this value as being morerepresentative of true ch806 clearance. The pharmacokinetic values forch806 are comparable to other chimeric antibodies reported to date(Steffens et al., 1997; Scott et al., 2001), and supports a weeklydosing schedule of ch806.

The quantitative dosimetry and pharmacokinetic results indicate thatthere is no saturable normal tissue compartment for ch806 for the doselevels assessed in this trial. Importantly, the lack of dose dependenceon pharmacokinetic and whole body and liver organ clearance is in markedcontrast to all reported studies of antibodies to wtEGFR (Baselga J. andArtega C. L. (2005) J. Clin. Oncol. 23, 2445-2449; Divgi et al. J. Natl.Cancer Inst. 83(2), 97-104; Baselga J (2001) Eur. J. Cancer 37 Suppl. 4,S16-22; Gibson et al. (2006) Clin. Colorectal Cancer 6(1), 29-31;Rowinsky et al. (2004) J. Clin. Oncol. 22, 3003-3015; Tan et al. (2006)Clin. Cancer Res. 12(21), 6517-6522) supporting the tumour specificityand lack of normal tissue binding of ch806 in humans. These observationsprovide compelling evidence of the potential for ch806 (or humanizedforms) to selectively target EGFR in tumor, avoid the normal toxicity ofother EGFR antibodies and kinase inhibitors (particularly skin)(Lacouture A E (2006) Nature Rev. Cancer 6, 803-812; Adams G. P. andWeiner L. M. (2005) Nat. Biotechnol. 23(9), 1147-1157) and potentiallyachieve greater therapeutic effect. Moreover, the possibility of payloaddelivery (due to the rapid internalisation of mAb 806 in tumor cells),and combination treatment with other biologics such as EGFR antibodiesand tyrosine kinase inhibitors where combined toxicity is likely beminimised, is strongly supported by the data from this trial. This studyprovides clear evidence of the ability to target an epitope on EGFR thatis specific for tumor, and further clinical development of this uniqueapproach to cancer therapy is ongoing.

EXAMPLE 27 Combination Treatment of Tumor Xenografts with hu806 andRadiation

The effect of hu806 in combination with radiation on tumor growth wasevaluated on subcutaneous A431 xenograft tumors implanted in nude mice.The sequence of hu806 used in this Example and in Examples 28-33included a change of the boxed cysteine residue shown in FIG. 55A to aleucine residue.

Briefly, 3×10⁶ cells were inoculated subcutaneously into the right hindflank of female nude mice. Tumors were allowed to establish for 6 days,at which point tumor volume was determined using electronic calipermeasurements. Tumor size was calculated using the formula: L×W²/2. Micewere allocated into treatment groups (n=10 per group) so that eachcohort of animals had equivalent mean tumor volume prior to initiationof therapy (approximately 170 mm³).

Animals were then dosed intraperitoneally with hu806 at 40 mg/kg threetimes a week for two weeks (total of 6 doses) and administered a single20 Gy dose of radiation localized to the tumor on the first day of hu806treatment. The hu806 and human IgG (control) dosing schedule isindicated by upper arrows in FIG. 99; administration of the single doseof radiation is indicated by the lower arrow in FIG. 99. Tumor volumewas measured on average twice a week for the duration of the experimentuntil the mean tumor volume in each group reached an endpoint of ≦3,000mm³.

As illustrated in FIG. 99, combination hu806 and radiation treatment(solid squares) was more effective at reducing mean tumor volume thaneither radiation or hu806 alone (triangles and upside down triangles,respectively).

EXAMPLE 28 Combination Treatement of Tumor Xenografts with hu806 andBevacizumab

The effect of hu806 in combination with Bevacizumab on tumor growth wasevaluated on subcutaneous A431 xenograft tumors implanted in nude mice.

Briefly, 3×10⁶ cells were inoculated subcutaneously into the right hindflank of female nude mice. Tumors were allowed to establish for 7 days,at which point tumor volume was determined using electronic calipermeasurements. Tumor size was calculated using the formula: L×W²/2. Micewere allocated into treatment groups (n=10 per group) so that eachcohort of animals had equivalent mean tumor volume prior to initiationof therapy (approximately 170 mm³).

Animals were then dosed intraperitoneally with hu806 at 40 mg/kg andBevacizumab at 2 mg/kg three times a week for two weeks (total of 6doses for each antibody). The hu806, Bevacizumab and human IgG (control)dosing schedule is indicated by arrows in FIG. 100. Tumor volume wasmeasured on average twice a week for the duration of the experimentuntil the mean tumor volume in each group reached an endpoint of ≦3,000mm³.

As illustrated in FIG. 100, combination hu806 and Bevacizumab treatment(solid squares) was more effective at reducing mean tumor volume thaneither Bevacizumab or hu806 alone (triangles and upside down triangles,respectively).

EXAMPLE 29 Combination Treatement of Tumor Xenografts with hu806 andCetuximab

The effect of hu806 in combination with Cetuximab on tumor growth wasevaluated on subcutaneous A431 xenograft tumors implanted in nude mice.

Briefly, 3×10⁶ cells were inoculated subcutaneously into the right hindflank of female nude mice. Tumors were allowed to establish for 7 days,at which point tumor volume was determined using electronic calipermeasurements. Tumor size was calculated using the formula: L×W²/2. Micewere allocated into treatment groups (n=10 per group) so that eachcohort of animals had equivalent mean tumor volume prior to initiationof therapy (approximately 170 mm³).

Animals were then dosed intraperitoneally with hu806 at 10 mg/kg andwith Cetuximab at 10 mg/kg three times a week for two weeks (total of 6doses for each antibody). The hu806, Cetuximab and human IgG (control)dosing schedule is indicated by arrows in FIG. 101. Tumor volume wasmeasured on average twice a week for the duration of the experimentuntil the mean tumor volume in each group reached an endpoint of ≦3,000mm³.

As illustrated in FIG. 101, combination hu806 and Cetuximab treatment(solid squares) was more effective at reducing mean tumor volume thaneither Cetuximab or hu806 alone (triangles and upside down triangles,respectively).

EXAMPLE 30 Combination Treatement of Tumor Xenografts with hu806 andErlotinib

The effect of hu806 in combination with Erlotinib on tumor growth wasevaluated on subcutaneous A431 xenograft tumors implanted in nude mice.

Briefly, 3×10⁶ cells were inoculated subcutaneously into the right hindflank of female nude mice. Tumors were allowed to establish for 4 days,at which point tumor volume was determined using electronic calipermeasurements. Tumor size was calculated using the formula: L×W²/2. Micewere allocated into treatment groups (n=10 per group) so that eachcohort of animals had equivalent mean tumor volume prior to initiationof therapy (approximately 160 mm³).

Animals were then dosed intraperitoneally with hu806 at 40 mg/kg threetimes a week for two weeks (total of 6 doses) and orally with Erlotinibat 25 mg/kg two times a day for 12 days. The hu806 and human IgG(control) dosing schedule is indicated by vertical arrows in FIG. 102;the Erlotinib dosing schedule is indicated by horizontal arrow in FIG.102. Tumor volume was measured on average twice a week for the durationof the experiment until the mean tumor volume in each group reached anendpoint of ≦3,000 mm³. [0777] As illustrated in FIG. 102, combinationhu806 and Erlotinib treatment (solid squares) was more effective atreducing mean tumor volume than either Erlotinib or hu806 alone(triangles and upside down triangles, respectively).

EXAMPLE 31 Combination Treatement of Tumor Xenografts with hu806 and 5FU

The effect of hu806 in combination with 5FU (fluorouracil) on tumorgrowth was evaluated on subcutaneous model SCC-15 xenograft tumorsimplanted in SCID (severely combined immunodeficient) Beige mice.

Briefly, 1×10⁶ cells were inoculated subcutaneously into the right hindflank of female SCID Beige mice. Tumors were allowed to establish for 11days, at which point tumor volume was determined using electroniccaliper measurements. Tumor size was calculated using the formula:L×W²/2. Mice were allocated into treatment groups (n=10 per group) sothat each cohort of animals had equivalent mean tumor volume prior toinitiation of therapy (approximately 210 mm³).

Animals were then dosed intraperitoneally with hu806 at 10 mg/kg threetimes a week for two weeks (total of 6 doses) and intraperitoneally with5FU at 50 mg/kg (with leucovorin at 15 mg/kg) on 2 consecutive daysbeginning 1 day after the 1^(st) and the 4^(th) hu806 dose (total of 4doses). The hu806 and human IgG (control) dosing schedule is indicatedby upper arrows in FIG. 103; the 5FU dosing schedule is indicated bylower arrows in FIG. 103. Tumor volume was measured on average twice aweek for the duration of the experiment until the mean tumor volume ineach group reached an endpoint of ≦3,000 mm³.

As illustrated in FIG. 103, combination hu806 and 5FU treatment (solidsquares) was more effective at reducing mean tumor volume than either5FU or hu806 alone (triangles and upside down triangles, respectively).

EXAMPLE 32 Combination Treatement of Tumor Xenografts with hu806 andCisplatin

The effect of hu806 in combination with Cisplatin on tumor growth wasevaluated on subcutaneous HNSCC SCC-15 xenograft tumors implanted inSCID Beige mice.

Briefly, 1×10⁶ cells were inoculated subcutaneously into the right hindflank of female SCID Beige mice. Tumors were allowed to establish for 11days, at which point tumor volume was determined using electroniccaliper measurements. Tumor size was calculated using the formula:L×W²/2. Mice were allocated into treatment groups (n=10 per group) sothat each cohort of animals had equivalent mean tumor volume prior toinitiation of therapy (approximately 210 mm³).

Animals were then dosed intraperitoneally with hu806 at 10 mg/kg threetimes a week for two weeks (total of 6 doses) and intravenously withCisplatin at 5 mg/kg on the initial day hu806 dosing (total of 1 dose).The hu806 and human IgG (control) dosing schedule is indicated by upperarrows in FIG. 104; the Cisplatin dosing schedule is indicated by lowerarrows in FIG. 104. Tumor volume was measured on average twice a weekfor the duration of the experiment until the mean tumor volume in eachgroup reached an endpoint of ≦3,000 mm³.

As illustrated in FIG. 104, combination hu806 and Cisplatin treatment(solid squares) was more effective at reducing mean tumor volume thaneither Cisplatin or hu806 alone (triangles and upside down triangles,respectively).

EXAMPLE 33 Combination Treatement of Tumor Xenografts with hu806, 5FUand Cisplatin

The effect of hu806 in combination with 5FU and Cisplatin on tumorgrowth was evaluated on subcutaneous HNSCC SCC-15 xenograft tumorsimplanted in SCID Beige mice.

Briefly, 1×10⁶ cells were inoculated subcutaneously into the right hindflank of female SCID Beige mice. Tumors were allowed to establish for 11days, at which point tumor volume was determined using electroniccaliper measurements. Tumor size was calculated using the formula:L×W²/2. Mice were allocated into treatment groups (n=10 per group) sothat each cohort of animals had equivalent mean tumor volume prior toinitiation of therapy (approximately 210 mm³).

Animals were then dosed intraperitoneally with hu806 at 10 mg/kg threetimes a week for two weeks (total of 6 doses); were dosedintraperitoneally with 5FU at 12.5 mg/kg or 25 mg/kg (with leucovorin at7.5 mg/kg or 15 mg/kg, respectively) on 2 consecutive days beginning 1day after the 1^(st) and the 4^(th) hu806 dose (total of 4 doses); andwere dosed intravenously with Cisplatin at 2.5 kg/mg or 5 kg/mg on theday of the 1^(st) and 4^(th) hu806 dose (total of 2 doses). The hu806and human IgG (control) dosing schedule is indicated by the upper row ofarrows in FIG. 105; the 5FU dosing schedule is indicated by the middlerow of arrows in FIG. 105; the Cisplatin dosing schedule is indicated bythe lower row arrows in FIG. 105. Tumor volume was measured on averagetwice a week for the duration of the experiment until the mean tumorvolume in each group reached an endpoint of ≦3,000 mm³. Results areshown in FIG. 7.

As illustrated in FIG. 105, combination hu806, 5FU and Cisplatintreatment (solid circles and solid upside down triangles) was moreeffective at reducing mean tumor volume than either Cisplatin incombination with 5FU (open circles and open upside down triangles) orhu806 alone (solid squares).

EXAMPLE 34 Sequence Comparisons

The VH chain and VL chain CDRs for each of mAb806, mAb175, mAb124,mAb1133, and hu806 are set forth and compared herein.

TABLE 19 Murine Antibody Isotype and CDR Sequence Comparisons (Kabat)¹CDR1 CDR2 CDR3 A. Variable Light Chain  806 (IgG2b) HSSQDINSNIG HGTNLDD  VQYAQFPWT  (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) 124 (IgG2a) HSSQDINSNIG  HGTNLDD  VQYGQFPWT  (SEQ ID NO: 28)(SEQ ID NO: 29) (SEQ ID NO: 30)  175 (IgG2a) HSSQDISSNIG  HGTNLED VQYGQFPWT  (SEQ ID NO: 135) (SEQ ID NO: 136) (SEQ ID NO: 137)1133 (IgG2a) HSSQDINSNIG  HGTNLDD  VQYGQFPWT  (SEQ ID NO: 38)(SEQ ID NO: 39) (SEQ ID NO: 40) B. Variable Heavy Chain  806 (IgG2b)SDFAWN  YISYSGNTRYNPSLKS  VTAGRGFPY  (SEQ ID NO: 15) (SEQ ID NO:1 6)(SEQ ID NO: 17)  124 (IgG2a) SDYAWN  YISYSANTRYNPSLKS ATAGRGFPY (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25)  175 (IgG2a) SDYAWN YISYSANTRYNPSLKS ATAGRGFPY  (SEQ ID NO: 130) (SEQ ID NO: 131)(SEQ ID NO: 132) 1133 (IgG2a) SDYAWN  YISYSGNTRYNPSLRS ATAGRGFPY (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35) ¹differences to themAb806 CDR sequences are underlined

The CDRs given above for the respective antibody isotypes are based on aKabat analysis. As will be apparent to those of skill in the art, theCDRs may also be defined based on other analysis, for example acomposite of Kabat and Chothia definitions. For example, applying acomposite Kabat and Chothia analysis to the above isotypes, thesequences of the VL chain CDRs and VH chains CDRs for the respectiveisotypes are as set forth in Table 27.

TABLE 20  Murine Antibody Isotype and CDR Sequence Comparisons (Composite Kabat and Chothia)¹ CDR1 CDR2 CDR3 A. Variable Light Chain 806 (IgG2b) HSSQDINSNIG   HGTNLDD  VQYAQFPWT  (SEQ ID NO: 18)²(SEQ ID NO: 139)² (SEQ ID NO: 20)²  124 (IgG2a) HSSQDINSNIG  HGTNLDD VQYGQFPWT  (SEQ ID NO: 28) (SEQ ID NO: 140) (SEQ ID NO: 30)  175 (IgG2a)HSSQDISSNIG   HGTNLED  VQYGQFPWT  (SEQ ID NO: 135) (SEQ ID NO: 141)(SEQ ID NO: 137) 1133 (IgG2a) HSSQDINSNIG  HGTNLDD  VQYGQFPWT (SEQ ID NO: 38) (SEQ ID NO: 142) (SEQ ID NO: 40) B. Variable Heavy Chain 806 (IgG2b) GYSITSDFAWN  GYISYSGNTRYNPSLKS  VTAGRGFPY (SEQ ID NO: 143)³ (SEQ ID NO: 144)³ (SEQ ID NO: 17)³  124 (IgG2a)GYSITSDYAWN  GYISYSANTRYNPSLKS  ATAGRGFPY  (SEQ ID NO: 145)(SEQ ID NO: 146) (SEQ ID NO: 25)  175 (IgG2a) GYSITSDYAWN GYISYSANTRYNPSLKS  ATAGRGFPY  (SEQ ID NO: 147) (SEQ ID NO: 148)(SEQ ID NO: 132) 1133 (IgG2a) GYSITSDYAWN  GYISYSGNTRYNPSLRS  ATAGRGFPY (SEQ ID NO: 149) (SEQ ID NO: 150) (SEQ ID NO: 35) ¹differences to themAb806 CDR sequences are underlined ²See FIG. 17 of U.S. patentapplication No. 10/145,598 (U.S. Pat. No. 7,589,180) ³See FIG. 16 ofU.S. patent application No. 10/145,598 (U.S. Pat. No. 7,589,180)

TABLE 21 mAb806 and hu806 CDR Sequence Comparisons (Kabat)¹ CDR1 CDR2CDR3 A. Variable Light Chain mAb806 HSSQDINSNIG  HGTNLDD  VQYAQFPWT(SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) hu806 HSSQDINSNIG HGTNLDD  VQYAQFPWT (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51)B. Variable Heavy Chain mAb806 SDFAWN  YISYSGNTRYNPSLKS VTAGRGFPY(SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) hu806 SDFAWN YISYSGNTRYQPSLKS VTAGRGFPY (SEQ ID NO: 44) (SEQ ID NO: 45)(SEQ ID NO: 46) ¹differences to the mAb806 CDR sequences are underlined

As shown above, the CDR sequences of mAb806, mAb175, mAb124 and mAb1133isotypes are identical except for highly conservative amino acid changesthat would be expected to give rise to homologous protein folding forepitope recognition. This data, cumulatively with the binding and otherdata provided in the Examples above, shows that these isotypes and thehu806 are closely-related family member variants exhibiting the sameunique properties discussed above for mAb806 (e.g., binding to anepitope on the EGFR that is accessible to binding only in overexpressed,mutated or ligand activated forms of the EGFR, resulting in uniquespecificity for tumor-expressed EGFR, but not wtEGFR in normal tissue)and demonstrating that antibodies of distinct variable region sequences,particularly of varying CDR sequences, have the same characteristics andbinding capabilities.

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrated and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

Various references are cited throughout the Specification and providedin a list of references above, each of which is incorporated herein byreference in its entirety.

1. A pharmaceutical composition comprising: (1) a first therapeuticallyactive agent, comprising an isolated antibody capable of binding EGFR ontumor cells that overexpress EGFR, and on tumor cells that express thetruncated version of the EGFR receptor, de2-7 EGFR, wherein saidantibody does not bind to the de2-7 EGFR junctional peptide consistingof the amino acid sequence of SEQ ID NO:13, wherein said antibody bindsto an epitope within the sequence of residues 287-302 of human wild-typeEGFR; and (2) a second therapeutically active agent.
 2. A pharmaceuticalcomposition according to claim 1, wherein said isolated antibodycomprises a heavy chain and light chain, wherein the variable region ofsaid heavy chain comprises polypeptide binding domain regionscorresponding to amino acids 26-36, 50-65, and 97-105 of SEQ ID NO: 11,and wherein the variable region of said light chain comprisespolypeptide binding domain regions corresponding to amino acids 24-34,50-56, and 89-97 of SEQ ID NO:
 12. 3. A pharmaceutical compositionaccording to claim 1, wherein said isolated antibody is selected fromthe group consisting of: an isolated antibody comprising a heavy chainand a light chain, wherein the variable region of said heavy chaincomprises polypeptide binding domain regions having the amino acidsequences set forth in SEQ ID NOS:23, 24, and 25, and wherein thevariable region of said light chain comprises polypeptide binding domainregions having amino acid sequences set forth in SEQ ID NOS:28, 29, and30; an isolated antibody comprising a heavy chain and a light chain,wherein the variable region of said heavy chain comprises polypeptidebinding domain regions having the amino acid sequences set forth in SEQID NOS:33, 34, and 35, and wherein the variable region of said lightchain comprises polypeptide binding domain regions having amino acidsequences set forth in SEQ ID NOS:38, 39, and 40; and an isolatedantibody comprising a heavy chain and a light chain, wherein thevariable region of said heavy chain comprises polypeptide binding domainregions having the amino acid sequences set forth in SEQ ID NOS:130,131, and 132, and wherein the variable region of said light chaincomprises polypeptide binding domain regions having amino acid sequencesset forth in SEQ ID NOS:135, 136, and
 137. 4. A pharmaceuticalcomposition according to claim 1, wherein said isolated antibodycomprises a heavy chain and a light chain, wherein the variable regionof said heavy chain comprises polypeptide binding domain regions havingthe amino acid sequences set forth in SEQ ID NOS:44, 45, and 46, andwherein the variable region of said light chain comprises polypeptidebinding domain regions having amino acid sequences set forth in SEQ IDNOS:49, 50, and
 51. 5. A pharmaceutical composition according to one ofclaims 2-4, wherein said second therapeutically active agent is ananti-cancer agent.
 6. A pharmaceutical composition according to claim 5,wherein said anti-cancer agent is selected from the group consisting oferlotinib, 5-fluorouracil, cisplatin, a combination of 5-fluorouraciland cisplatin, bevacizumab, and cetuximab.
 7. A pharmaceuticalcomposition according to claim 5, wherein said anti-cancer agent is atyrosine kinase inhibitor.
 8. A pharmaceutical composition according toclaim 7, wherein said tyrosine kinase inhibitor is selected from thegroup consisting of AG1478, ZD1839,STI571, OSI-774, SU-6668, andcombinations thereof.
 9. A pharmaceutical composition according to claim5, wherein said anti-cancer agent is an anti-EGFR antibody.
 10. Apharmaceutical composition according to claim 9, wherein said anti-EGFRantibody is selected from the group consisting of the anti-EGFRantibodies 528, SC-03,DR8. 3, L8A4, Y10, ICR62, ABX-EGF, andcombinations thereof.
 11. A pharmaceutical composition according toclaim 5, wherein said anti-cancer agent is selected from the groupconsisting of: 4-desacetylvinblastine-3-carbohydiazide;5-fluoro-2′-deoxyuridine; 5-fluorouracil decarbonizes; 6-mercaptopurine;6-thioguanine; abrin; abrin A chain; actinomycin D; actinomycin D,1-dehydrotestosterone; adriamycin; alkylating agents;alkylphosphocholines; aminopterin; angiogenin; angiostatin;anthracyclines; anthramycin; anti-angiogenics; anti-folates;anti-metabolites; anti-mitotics; antibiotics; ara-C; auristatinderivatives; auristatin E; auristatin E valeryl benzylhydrazone;auristatin F phenylene diamine; auristatins; auromycins; bis-iodo-phenolmustard; bismuth; bleomycin; busulfan; calicheamicin; carboplatin;carminomycin; carmustine; cc-1065 compounds; chlorambucil; colchicin(colchicine); combrestatin; crotin; curicin; cyclothosphamide;cytarabine; cytochalasin B; cytosine arabinoside; cytoxin; dacarbazine;dactinomycin (actinomycin); daunorubicin (daunomycin); dibromomannitol;dihydroxy anthracin dione; diphtheria toxin; dolastatin-10; doxetaxel;doxorubicin; doxorubicin hydrazides; duocarmycins; emetine; endostatin;enediyenes; enomycin; epirubicin; esperamicin compounds; ethidiumbromide; etoposide; gelonin; glucocorticoids; gramicidin D; granulocytecolony stimulating factor; granulocyte macrophage colony stimulatingfactor; idarubicin; intercalating agents; interleukin-1; interleukin-2;interleukin-6; lidocaine; lomustine; lymphokine; maytansinols;mechlorethamine; melphalan (and other related nitrogen mustards);methotrexate; minor groove-binders; mithramycin; mitogellin; mitomycinC; mitomycins; mitoxantrone; MMAF-dimethylaminoethylamine;MMAF-N-t-butyl; MMAF-tetraethylene glycol; modeccin A chain; mono-methylauristatin E (MMAE); mono-methyl auristatin F (MMAF);morpholinodoxorubicin; N2′-deacetyl-N2′-(c-mercapto-1oxopropyl)-maytansine (DM1);N2′-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4);neocarzinostatin; nerve growth factor (and other growth factors);onapristone; paclitaxel; PE40; phenomycin; platelet derived growthfactor; prednisone; procaine; propranolol; Pseudomonas exotoxin A;puromycin; radioactive isotopes (such as, for example and withoutlimitation, At211, Bi212, Bi213, Cf252, I125, I131, In111, Ir192, Lu177,P32, Re186, Re188, Sm153, Y90, and W188); retstrictocin; ricin A;ricins; Sapaonaria officinalis inhibitor; saporin; streptozotocin;suramin; tamoxifen; taxanes; taxoids; taxol; tenoposide; tetracaine;thioepa chlorambucil; thiotepa; thrombotic agents; tissue plasminogenactivator; topoisomerase I inhibitors; topoisomerase II inhibitors;toxotere; tumor necrosis factor; vinblastine; vinca alkaloids; vincas;vincristine; vindesine; vinorelbine; yttrium; α-interferon; α-sarcin;and β-interferon.
 12. A method for the treatment of cancer in mammals,comprising administering to a mammal a therapeutically effective amountof (1) an isolated antibody capable of binding EGFR on tumor cells thatoverexpress EGFR, and on tumor cells that express the truncated versionof the EGFR receptor, de2-7 EGFR, wherein said antibody does not bind tothe de2-7 EGFR junctional peptide consisting of the amino acid sequenceof SEQ ID NO:13, wherein said antibody binds to an epitope within thesequence of residues 287-302 of human wild-type EGFR; and (2) one ormore doses of radiation.
 13. A method for the treatment of cancer inmammals, comprising administering to a mammal a therapeuticallyeffective amount of a pharmaceutical composition according to claim 1.14. A method for the treatment of cancer in mammals according to claim12 or 13, wherein said isolated antibody is selected from the groupconsisting of: an isolated antibody comprising a heavy chain and lightchain, wherein the variable region of said heavy chain comprisespolypeptide binding domain regions corresponding to amino acids 26-36,50-65, and 97-105 of SEQ ID NO: 11, and wherein the variable region ofsaid light chain comprises polypeptide binding domain regionscorresponding to amino acids 24-34, 50-56, and 89-97 of SEQ ID NO: 12;an isolated antibody comprising a heavy chain and a light chain, whereinthe variable region of said heavy chain comprises polypeptide bindingdomain regions having the amino acid sequences set forth in SEQ IDNOS:23, 24, and 25, and wherein the variable region of said light chaincomprises polypeptide binding domain regions having amino acid sequencesset forth in SEQ ID NOS:28, 29, and 30; an isolated antibody comprisinga heavy chain and a light chain, wherein the variable region of saidheavy chain comprises polypeptide binding domain regions having theamino acid sequences set forth in SEQ ID NOS:33, 34, and 35, and whereinthe variable region of said light chain comprises polypeptide bindingdomain regions having amino acid sequences set forth in SEQ ID NOS:38,39, and 40; and an isolated antibody comprising a heavy chain and alight chain, wherein the variable region of said heavy chain comprisespolypeptide binding domain regions having the amino acid sequences setforth in SEQ ID NOS:130, 131, and 132, and wherein the variable regionof said light chain comprises polypeptide binding domain regions havingamino acid sequences set forth in SEQ ID NOS:135, 136, and
 137. 15. Amethod for the treatment of cancer in mammals according to claim 14,wherein said anti-cancer agent is selected from the group consisting oferlotinib, 5-fluorouracil, cisplatin, a combination of 5-fluorouraciland cisplatin, bevacizumab, and cetuximab.
 16. A method for thetreatment of cancer in mammals according to claim 12 or 13, wherein saidcancer is a brain-resident cancer that produces aberrantly expressedEGFR.
 17. A method for the treatment of cancer in mammals according toclaim 16, wherein said brain-resident cancer is selected from the groupconsisting of glioblastomas, medulloblastomas, meningiomas, neoplasticastrocytomas and neoplastic arteriovenous malformations.